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Archive for August, 2012
My estimation on the Seahawks 53 man roster
This is my complete projected Seahawks 53 man roster. This started as a piece I was asked to do for Northwest Sports Beat. That piece called for me to select 5 players who would stay and 5 who would go. Like an idiot, I decide the only way to do it properly was to break down the entire 53 man roster.
This will simply be a list to acts as an addition to the post on Northwest Sports Beat where I actually break down some of the more difficult decisions.
Offense (26)
Quarterback (3)
Russell Wilson
Matt Flynn
Josh Portis- Released
Running Back (4)
Marshawn Lynch
Michael Robinson
Robert Turbin
Kregg Lumkin
Leon Washington- Kept
Wide Receiver (6)
Sidney Rice
Braylon Edwards
Doug Baldwin
Golden Tate
Ben Obomanu
Charly Martin
Tight End (3)
Zach Miller
Kellen Winslow
Anthony McCoy
Offensive Line (10)
Russell Okung
Max Unger
John Moffitt
Breno Giacomini
Paul McQuistan
Lemuel Jeanpierre
Paul Fanaika- released
JR Sweezy
Allen Barbre- suspended list
Frank Omlyale
James Carpenter- Activated from PUP
Defense (24)
Cornerback (5)
Brandon Browner
Richard Sherman
Jeremy Lane
Byron Maxwell
Marcus Trufant
Safety (4)
Earl Thomas
Kam Chancellor
Jeron Johnson
Chris Maragos
Winston Guy- Kept
Defensive Line (9)
Red Bryant
Chris Clemons
Brandon Mebane
Bruce Irvin
Jason Jones
Greg Scruggs
Allen Branch
Jaye Howard
Clinton McDonald
Linebacker (6)
Bobby Wagner
Leroy Hill
KJ Wright
Mike Morgan
Malcolm Smith
Heath Farewell
Special Teams (3)
Clint Gresham
Steven Hauschka
Jon Ryan
Wether you like my decisions or you hate them, please take the time to read the reasoning behind some of them on Northwest Sports Beat and let me know how you feel.
Your new Seahawks starting QB Russell Wilson: love 'em & hate 'em
Pete & John announce Wilson starter
Some people are in love with the idea of having Russell Wilson start for the Seahawks, others hate the idea. Personally, I fall somewhere in the middle.
Normally I do the Love 'em section followed by the Hate 'em section, but I feel in order to get the season started on the right foot, I am going to end this piece on a high note.
Hate 'em (Hate is too strong a word)
Throughout the whole "quarterback competition" it was called a "3 player race" yet I felt like the position wasMatt Flynn's job to lose. Matt Flynn played very well, every-time he saw live action.
The kid gloves are off says Pete Carroll
This isn't a matter of me being right or me being wrong, this is a matter of conventional wisdom. Sure, there have been many quarterbacks who have played in their rookie season and done very well, but can you name one that was not forced into the starting job? More…
Five Seahawks who impressed against the Chiefs Friday night
Nice Game Boys! Impressive!
Seattle 44, Kansas City 14
When: 5:00 PST, Friday, August 24, 2012
Where: Arrowhead Stadium, Kansas City, Missouri
Temperature: 90
Dominate: "To rule; govern; control"…
That one word sums up the Seahawks-Chiefs game on Friday. There was not one instance, not one aspect, of the game when the team that controlled the game was at question. In every aspect of the game, the Seattle Seahawks DOMINATED the Kansas City Chiefs.
It has been difficult to select the five guys to write about in these posts each week, this week was no exception. Like every week, those chosen are ordered by jersey number.
Russell Wilson #3
Seahawks player grades vs Broncos: Defensive backs
Seahawks defensive back grades
Like I did with the receivers, I am going to just put the grades on this post unless I am asked to go into detail. Thank you all for your patience.
Richard Sherman #25
last week: C
this week: B
Brandon Browner #39
last week: A-
this week: B
Marcus Trufant #23
last week: C
this week: B
Jeremy Lane #1
last week: b
this week: C
Coye Francies #37
last week: c+
this week: C-
Phillip Adams #35
last week: f
this week: d+
Jeron Johnson #32
Last week: c
this week: B+
Kam Chancellor #31
last week: C+
this week: B
Chris Maragos #42
last week: c
this week: B
Earl Thomas #29
Last week: B+
this week: C
Winston Guy #27
last week: c-
this week: d-
Seahawks player grades vs Broncos: Receivers
Seahawks wide receiver/tight ends grades
We are in the 11th hour before tonights Seahawks-Cheifs matchup. With that in mind, I am going to give players their grades without breaking down why. If there are any players that you would like to see the reasonings behind the grade I gave just let me know, I will go back and add that in.
Wide Receiver
Jermaine Kearse #8
last week: c+
this week: b
Deon Butler #11
last week: b
this week: B-
Charly Martin #14
last week: B+
this week: b-
Braylon Edwards #17
last week: b-
this week: C+
Phil Bates #13
the grade: C+
Kris Durham: #16
last week: C
this week: C+
Lavasier Tiunei #19
last week: b-
this week: C+
Golden Tate #81
last week: C
this week: C
Ricardo Lockette #83
the grade: D+
Terrell Owens #10
the grade: F
Tight End
Kellen Winslow #82
the grade: B
Anthony McCoy #85
Last week: C+
This week: B+
Cameron Morrah #88
last week: F
this week: C+
Sean McGrath #45
Last week: C+
this week: C+
Cooper Heifet #84
the grade: C-
Seahawks player grades vs Broncos: Defensive line
Seahawks player grades
This is the 4th part of the Seahawks player grades from the Broncos game. Already posted are; Quarterback, running back and linebacker.
In order to keep this post manageable I am going to be very brief in the descriptions. If you would like me to elaborate on any player in particular let me know and I will happily add that.
Defensive Line
Brandon Mabane #92
Last week Brandon received a B
Brandon Mebane is a solid player and he drew several double teams against the Broncos. He was strong in the running game as usual and he was also able to create some pressure on the quarterback. Mabane does need to work on his backside contain.
the grade: A
Pep Levingston #93
Last week he received a C
Pep Levingston is a very athletic guy and it is not surprising that he is able to get pressure on the quarterback, or his ability to pursue plays from the backside. What surprises me is his ability to hold the point of attack and the strength he shows when he throws guards to the ground to get run penetration.
the grade: A-
Red Bryant #79
Last week Big Red Bryant received a C+
We all know how solid Red Bryant is in the running game, but in years past he has been a liability in the passing game. This year he is paying more attention to the quarterback throwing the ball and working to get his hands up to deflect it. Against the Broncos he was able to tip a pass at the line that KJ Wright intercepted. Conditioning may be a question, he looks a little more chubby than he has in years past and he seemed to take a couple plays off.
the grade: B+
Clinton McDonald #69
Last weeks Clinton received a C
Denver's offense showed Clinton McDonald a lot of respect on Saturday. They spent a lot of his snaps double teaming him, yet he was still able to get some run penetration. I dinged him a bit on his grade because he definitely took one play off, he didn't even pretend to defend on the play. He should have been rushing the passer but just engaged the blocker and stood around.
the grade: B
Dexter Davis (Waived/Injured)
Unfortunately for Dexter Davis, he injured his hip during the game against the Broncos and the Seahawks parted ways with him earlier in the week. I believe he was a long shot to make this years team had he stayed healthy.
the grade: B-
Greg Scruggs #98
Last week Greg really impressed me and received a B-
This week he was even more impressive making plays. Greg Scruggs has shown an ability to effect the passing game by collapsing the pocket while lining up at the defensive tackle position. Whether he was lined up inside at tackle or outside at defensive end he was able to create pressure, recording a sack while at DE. The one thing that is annoying me about his play is that far too often he is the last player off the snap.
the grade: b-
Bruce Irvin #51
Last week Bruce received a C+
This week I notice some signs of improvement in his pass rush. There were two plays that really stood out to me. When Bruce Irvin threw the tackle to the ground and when he pushed a tackle into Peyton Manning's face. That said, Bruce is still struggling in his pass rush, partially because he is getting a slow jump off that snap. I would expect that to correct itself as he becomes more comfortable in the defense. Players always play faster when they stop thinking so much and start playing. Bruce was a liability in the running game, especially when they run at him but that is to be expected from a guy his size.
the grade: C+
Alan Branch #99
Allen Branch was able to create a lot of pressure from the inside, on one play the Broncos triple teamed him, yet he still able to get pressure on the quarterback. When the offense ran the ball at him he was unable to hold the point of attack. Allen was pancaked on a pass rush attempt.
the grade: C+
Pierre Allen
Last week Pierre struggled and receive a unit low D-
This week Pierre Allen played a lot better than he did last week. He was able to get some pressure in the running game as well as the passing game. Allen's biggest play of the night was when he was able to get to the running back behind the line and bring him down. The worst play was when he was run out of the play 1:1 by a right guard.
the grade: C+
Jaye Howard #94
Last week Jaye Howard looked really good and received a B-
This week was a bit of a rough week for Jaye. He was able to get a quarterback pressure and a tackle for a loss. Other than those two plays Howard struggled to stay under control.
the grade: C-
Chris Clemons #91
Last week he received a C-
For the second straight week Chris Clemons under performed. Chris is losing 1:1 battles for the most part. I need to see a more consistent pass rush than Chris has been giving. Hopefully the money has not gone to his head, but if he doesn't turn it around soon I'm going to start becoming concerned that is what has happened.
the grade: D
Seahawks player grades vs Broncos: Running back
Seattle Seahawks player grades
Before I get into the individual players in this group, I would just like to say how fantastic the group was as an entire unit. I cannot remember having a position group where every player evaluated had a grade above a C before today.
Earlier this week I posted quarterback and linebacker grades.
Robert Turbin #22
Last week Robert was solid, but did not stand out and received a C for the week
the good:
One of the biggest holes on the team was at backup running back. When the Seahawks drafted Robert Turbin they hoped he could carry his running tough style from college over to the NFL. If he could run like he did in college at the NFL level the Seahawks would have a guy playing behind Marshawn Lynch who could run like Marshawn Lynch.
Don't get me wrong, his running style isn't identical to Marshawn's, but it is very similar. Robert Turbin is a tough runner and he is learning how to stick his cleat in the ground, make one cut and go. Of course he breaks a lot of tackles, but he also has a nice lateral jump cut that allows him to avoid tacklers.
the bad:
Like many rookies Robert Turbin has a lack of awareness that, at times, creates a liability on offense. No one expects him to be perfect. If he was he wouldn't have made it out of the first round.
the grade: A-
Kregg Lumpkin #20
Last week Kregg Lumpkin led all running backs with an A-
the good:
On the Seahawks first touchdown of the game Kregg Lumpking did a nice job getting to the outside by using his free hand to avoid the only defender he had to beat. Kregg also showcased a nice little cut to avoid tacklers, as well as a little power. In the beginning of the game he had a nice cut block to clear a passing lane.
the bad:
For the most part Lumpkin struggled in pass protection. He was beat a couple times that force Russell Wilson to scramble to avoid a sack.
the grade: A-
Marshawn Lynch #24
Last week Marshawn did not play
the good:
I don't really think I need to say anything other than, Marshawn looked like Marshawn out there. When he had the ball in his hands he did what he does.
the bad:
Marshawn Lynch was beat by a blitzer early in the game, but was smart enough to hold the defender and take the penalty rather than allowing his quarterback to take a big hit.
the grade: B
Tyrell Sutton #30
Last week Tyrell Sutton received a C-
the good:
Last week it looked like Tyrell Sutton was going to fade into obscurity, and allow Kregg Lumpkin to take the Seahawks 4th running back position. This week he looked much sharper. In particular the play that he made when he caught the screen pass for a touchdown, that play was all about individual effort. In order to score the touchdown Tyrell had to break 3 tackles. It reminded me of a play Leonard Weaver made back in the days when he was trying to earn a roster spot.
the bad:
Sutton needs to work on his pass blocking ability, specifically holding his blocks longer.
the grade: B
Leon Washington #33
Last week Leon received a B
the good:
Leon Washington did not see much playing time in the game but, in limited action, he was able to make a few plays that stood out. On one play he did a really good job picking up a blitzer and gave his quarterback the time to throw the ball. One thing that Leon does as well as anyone on the Seahawks roster is set up his blockers. He has the patience to give them time to get into position before he makes his move.
the bad:
The return game is really the time for Leon to shine, and in Denver Leon Washington made a stupid mistake. There is no reason to call for a fair catch around the 45 yard line and then let the ball hit the ground. Nine times out of ten that is going to put your offense much deeper in their own territory.
the grade: B-
Via Taua #40
Last week Via struggled early the game, but came on strong and received a B-. This week Via Taua played the entire game with Michael Robinson sidelined.
the good:
Via shows the ability to make some pretty big blocks look easy.
the bad:
He reminds me a little of the player Michael Robinson was a couple years ago. What I mean is, at times he goes for the big hit on the defender but doesn't stick his block. In the NFL defenders are very good at absorbing those hits and then making a play. Via Taua will need to make the same adjustments Robinson did if he ever wants to be a solid NFL fullback.


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subset: commoncrawl


text:
<REQUEST>Spotify Add-onn
Junior Member Posts: 1 Joined: Jan 2012 Reputation: 0 |
2012-02-18 20:48
Post: #1
Is there already a Spotify Add On ?
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Fan Posts: 533 Joined: Jan 2011 Reputation: 0 |
2012-02-18 22:59
Post: #2
would love to know too for ATV2 thanks all
find quote |
Junior Member Posts: 2 Joined: Mar 2012 Reputation: 0 |
2012-03-14 18:31
Post: #3
Didn't test it: http://forum.xbmc.org/showthread.php?tid=67012
find quote |
Fan Posts: 443 Joined: Aug 2008 Reputation: 4 Location: UK |
2012-03-16 16:33
Post: #4
The link given by Spatnonkl is for a whole separate build of XBMC which incorporates Spotify, rather than an Addon.
There is also an addon in development here: http://forum.xbmc.org/showthread.php?tid=125147 I haven't tried it, but the comments in the thread are generally positive. However, do bear in mind that it seems to be a work-in-progress, so don't expect it to be working flawlessly. NOTE: this Addon does not currently work on AppleTV2/iOS. The reason for this is given in the thread that I've linked to above. I'm really hoping this gets fixed because my primary instance of XBMC runs on an AppleTV, but I have no idea if/when it will get fixed. |
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Junior Member Posts: 7 Joined: May 2012 Reputation: 0 |
2012-05-14 13:22
Post: #5
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Junior Member Posts: 16 Joined: Jun 2011 Reputation: 1 Location: Apeldoorn, Netherlands |
2012-05-18 23:59
Post: #6
Samsung 50" 3D Plasma, I'm old school, like to do everything from one main pc, Core i7 930, Gigabyte GA-X58A-UD7, 2x ATi 5750, 1x SSD, 8TB HD total. XBMC with 3D subs working, SpotyXBMC2, XBMC Android remote, Sickbeard, SABnzbd+, Newsleecher. 60Mb Internet |
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text:
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text:
Page Contents
- Fixable Polar Tracers
- Nonfixable Polar Tracers
- Caged Fluorescent Dye Tracers
- Fluorescent Retrograde Tracers
- NeuroTrace Fluorescent Nissl Stains
- Polar Spin Label
- Signal Amplification of Polar Tracers
- Influx Pinocytic Cell-Loading Reagent
- Loading P2X7 Receptor–Expressing Cells
- Data Table
- Ordering Information
We prepare a wide variety of highly water-soluble dyes and other detectable probes that can be used as cell tracers. In most cases, the polarity of these water-soluble probes is too high to permit them to passively diffuse through cell membranes. Consequently, special methods for loading the dyes into cells must be employed, including microinjection, pinocytosis or techniques that temporarily permeabilize the cell's membrane (Techniques for loading molecules into the cytoplasm—Table 14.1). Our Influx pinocytic cell-loading reagent (I14402, see below and Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8) is particularly useful for loading many of the polar tracers in this section—as well as the dextrans and fluorescent proteins described in Fluorescent and Biotinylated Dextrans—Section 14.5 and Protein Conjugates—Section 14.7—into many types of cells. Permeabilization of cells with staphylococcal α-toxin or the saponin ester β-escin is reported to make the membrane of smooth muscle cells permeable to low molecular weight (<1000 daltons) molecules, while retaining high molecular weight compounds. Electroporation has been used to transport several of the polar tracers through the skin and into cells. Many of these tracers can also be loaded into cells noninvasively as their cell-permeant acetoxymethyl (AM) esters, which are discussed in more detail in Viability and Cytotoxicity Assay Reagents—Section 15.2.
Alexa Fluor Hydrazides and Hydroxylamines
Molecular Probes fluorescent hydrazide and hydroxylamine derivatives continues to expand (Reagents for Modifying Aldehydes and Ketones—Section 3.3, Molecular Probes hydrazine, hydroxylamine and amine derivatives—Table 3.2). The blue-fluorescent Alexa Fluor 350 hydrazide and Alexa Fluor 350 hydroxylamine (A10439, A30627), green-fluorescent Alexa Fluor 488 hydrazide and Alexa Fluor 488 hydroxylamine (A10436, A30629; ), orange-fluorescent Alexa Fluor 555 and Alexa Fluor 568 hydrazides (A20501MP, A10437, A10441; Figure 14.3.1), red-fluorescent Alexa Fluor 594 hydrazide (A10438, A10442) and far-red–fluorescent Alexa Fluor 633 hydrazide, Alexa Fluor 647 hydrazide and Alexa Fluor 647 hydroxylamine (A30634, A20502, A30632) are likely the best overall polar tracers in each of their various spectral ranges. These low molecular weight, cell membrane–impermeant molecules (Alexa Fluor 350 hydrazide, 349 daltons; Alexa Fluor 350 hydroxylamine, 585 daltons; ~570–760 daltons for the Alexa Fluor 488, 568 and 594 hydrazides and hydroxylamine; and about 1200 daltons for the Alexa Fluor 555 and 647 hydrazides and hydroxylamine) possess several properties that are superior to those of the widely used neuronal tracer lucifer yellow CH (L453, L682, L1177, L12926). Like lucifer yellow CH, the hydrazide moiety of the Alexa Fluor derivatives makes these tracers fixable by common aldehyde-based fixatives. We have determined that Alexa Fluor 594 hydrazide has a water solubility of ~84 mg/mL and the other Alexa Fluor hydrazides are likely to have comparable or higher water solubility.
Our rabbit polyclonal antibody to the Alexa Fluor 488 fluorophore (A11094, Anti-Dye and Anti-Hapten Antibodies—Section 7.4) quenches the fluorescence of the Alexa Fluor 488 dye (Anti–Lucifer Yellow Dye, Anti–Alexa Fluor 405/Cascade Blue Dye and Anti–Alexa Fluor 488 Dye Antibodies—Note 14.1) and, following cell fixation and permeabilization, can be used in conjunction with the reagents in our Tyramide Signal Amplification (TSA) Kits (TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2) to amplify the signal or with the anti–rabbit IgG conjugate of NANOGOLD or Alexa Fluor FluoroNanogold 1.4 nm gold clusters (N24916, A24926, A24927; Secondary Immunoreagents—Section 7.2) and the associated LI Silver Enhancement Kit (L24919, Secondary Immunoreagents—Section 7.2) for correlated fluorescence and light microscopy studies.
Although lucifer yellow CH can be used for confocal laser-scanning microscopy, its extinction coefficient at the 488 nm spectral line of the argon-ion laser (~700 cm-1M-1) is only about 1% of that of Alexa Fluor 488 hydrazide and Alexa Fluor 488 hydroxylamine (≥71,000 cm-1M-1) (Figure 14.3.2). Furthermore, the high photostability of the Alexa Fluor dyes permits their detection in very fine structures that cannot be seen with lucifer yellow CH staining. All of these Alexa Fluor dyes are remarkably bright and photostable. In addition, the Alexa Fluor hydrazide salts have high water solubility (typically greater than 8%). We offer the Alexa Fluor 568 and Alexa Fluor 594 hydrazides either as solids (A10437, A10438) or as 10 mM solutions in 200 mM KCl (A10441, A10442). The 10 mM solutions have been filtered through a 0.2 µm filter to remove any insoluble material prior to packaging. The Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 633 and Alexa Fluor 647 hydrazides (A10436, A20501MP, A30634, A20502) and Alexa Fluor 350, Alexa Fluor 488 and Alexa Fluor 647 hydroxylamines (A30627, A30629, A30632) are available only as solids. Our Alexa Fluor 350 hydrazide and Alexa Fluor 350 hydroxylamine, which are sulfonated coumarin derivatives (), are some of the few polar tracers that exhibit bright blue fluorescence.
Figure 14.3.1 Confocal image stack of a 10,000 MW Calcium Green dextran–labeled (C3713, Fluorescent Ca2+ Indicator Conjugates—Section 19.4) climbing fiber in a sagittal cerebellar slice, showing incoming axon and terminal arborization (in yellow). The Purkinje cell innervated by this climbing fiber was labeled with Alexa Fluor 568 hydrazide (A10437, A10441) via a patch pipette and visually identified using bright-field microscopy. Image contributed by Anatol Kreitzer, Department of Neurobiology, Harvard Medical School. |
Figure 14.3.2 Absorption spectra showing that the molar extinction coefficient (EC) at 488 nm of Alexa Fluor 488 hydrazide (A10436) in water (green line) is approximately 100-fold greater than that of lucifer yellow CH (L453, L682, L1177, L12926) in water (blue line). |
Other Alexa Fluor Derivatives
To allow amplification of signals, especially in the finer processes of dye-filled neurons, we also offer Alexa Fluor 488 biocytin (A12924), Alexa Fluor 546 biocytin (A12923) and Alexa Fluor 594 biocytin (A12922). These unique probes combine our Alexa Fluor 488, Alexa Fluor 546 and Alexa Fluor 594 fluorophores with biotin and an aldehyde-fixable primary amine (see "Fluorescent Biotin Derivatives," below). In addition, we offer the bright blue-fluorescent Alexa Fluor 405 cadaverine (A30675, see below) as well as several other Alexa Fluor cadaverines (Reagents for Modifying Aldehydes and Ketones—Section 3.3, Molecular Probes hydrazine, hydroxylamine and amine derivatives—Table 3.2), all of which should be useful as tracing molecules because they are exceptionally bright, small and water soluble, and they each contain an aldehyde-fixable functional group. Alexa Fluor 546 biocytin has been used to label streptavidin-coated particles in order to quantitate fluorescence signals in an automated imaging system designed for analyzing immobilized particle arrays.
Lucifer Yellow CH
Lucifer yellow CH (LY-CH or LY, ) has long been a favorite tool for studying neuronal morphology because it contains a carbohydrazide (CH) group that allows it to be covalently linked to surrounding biomolecules during aldehyde-based fixation. Loading of this polar tracer and other similar impermeant dyes is usually accomplished by microinjection, pinocytosis, scrape loading, ATP-induced permeabilization or osmotic shock (Techniques for loading molecules into the cytoplasm—Table 14.1), but can also be accomplished in cell suspensions or with adherent cells by using our Influx pinocytic cell-loading reagent (I14402, see below). Lucifer yellow CH localizes in the plant vacuole when taken up either through what is thought to be anion-transport channels or by fluid-phase endocytosis. Upon injection into the epidermal cells of Egeria densa leaves, lucifer yellow CH reportedly moved into the cytoplasm of adjacent cells, localized in the plant vacuole or moved in and out of the nucleus. The lithium salt of lucifer yellow CH is widely used for microinjection because of its relatively high water solubility (~8%). In addition to the solid (L453), we offer the lithium salt of lucifer yellow CH as a filtered 100 mM solution (L12926), ready for microinjection. The potassium salt (L1177, solubility ~1%) or the ammonium salt of lucifer yellow CH (L682, solubility ~6%) may be preferred in applications where lithium ions interfere with biological function.
Although its weak absorption at 488 nm (EC ~700 cm-1M-1) (Figure 14.3.2) makes it inefficiently excited with the argon-ion laser, lucifer yellow CH has been used as a neuronal tracer in some confocal laser-scanning microscopy studies. For electron microscopy studies, lucifer yellow CH can be used to photoconvert diaminobenzidine (DAB) into an insoluble, electron-dense reaction product (Fluorescent Probes for Photoconversion of Diaminobenzidine Reagents—Note 14.2). Alternatively, rabbit anti–lucifer yellow dye antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4) can be used in conjunction with our goat anti–rabbit IgG antibody conjugated to either NANOGOLD or Alexa Fluor FluoroNanogold 1.4 nm gold clusters (Secondary Immunoreagents—Section 7.2) and the LI Silver (LIS) Enhancement Kit (L24919, Secondary Immunoreagents—Section 7.2) to develop a more permanent, fade-free colorimetric or electron-dense signal from dye-filled neurons that is suitable for light or electron microscopy (Anti–Lucifer Yellow Dye, Anti–Alexa Fluor 405/Cascade Blue Dye and Anti–Alexa Fluor 488 Dye Antibodies—Note 14.1).
Intracellular injection of lucifer yellow CH has been extensively employed to delineate neuronal morphology in live neurons () and in fixed brain slices, as well as to investigate intercellular communication through gap junctions. Lucifer yellow CH can also be used to label neurons by using dye-filled electrodes during electrophysiological recording in order to correlate neuronal function with structure and connectivity.
Other Lucifer Yellow Derivatives
Like lucifer yellow CH, lucifer yellow ethylenediamine (A1339) is fixable with standard aldehyde-based fixatives and can be used as a building block for new lucifer yellow derivatives. The thiol-reactive lucifer yellow iodoacetamide (L1338) can also be used as a microinjectable polar tracer, as well as for preparing fluorescent liposomes and for detecting the accessibility of thiols in membrane-bound proteins. In addition to these lucifer yellow derivatives, we offer a lucifer yellow–conjugated 10,000 MW dextran (D1825, Fluorescent and Biotinylated Dextrans—Section 14.5).
Cascade Blue Hydrazide
Molecular Probes Cascade Blue hydrazide is a fixable analog of the nonfixable, bright blue-fluorescent tracer methoxypyrenetrisulfonic acid (MPTS). All of the Cascade Blue hydrazide derivatives have reasonable water solubility, ~1% for the sodium and potassium salts (C687, C3221) and ~8% for the lithium salt (C3239). They also exhibit a stronger absorption (EC400 nm >28,000 cm-1M-1) and quantum yield (~0.54 in water) than lucifer yellow CH. In addition, Cascade Blue derivatives have good photostability and emissions that are well resolved from those of fluorescein and lucifer yellow CH. Cascade Blue hydrazide, which readily passes through gap junctions, and lucifer yellow derivatives can be simultaneously excited at 405 nm (Figure 14.3.3) for two-color detection at about 430 and 530 nm. Cascade Blue dyes, lucifer yellow CH and sulforhodamine 101 can be used in combination for three-color mapping of neuronal processes (Figure 14.3.4). We also offer anti–Alexa Fluor 405/Cascade Blue dye antibodies (Anti-Dye and Anti-Hapten Antibodies—Section 7.4) for localizing Cascade Blue dye–filled cells following fixation (Anti–Lucifer Yellow Dye, Anti–Alexa Fluor 405/Cascade Blue Dye and Anti–Alexa Fluor 488 Dye Antibodies—Note 14.1). Like lucifer yellow CH, Cascade Blue hydrazide and some other polar tracers are taken up by plants and sequestered into their central vacuoles. In onion epidermal cells, this uptake of Cascade Blue hydrazide is blocked by probenecid (P36400, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8), indicating that transfer may be through anion-transport channels.
Figure 14.3.3 Absorption spectra for equal concentrations of Cascade Blue hydrazide (C687, C3221, C3239) and lucifer yellow CH (L453, L682, L1177, L12926) in water. |
Figure 14.3.4 Normalized fluorescence emission spectra for Cascade Blue hydrazide (C687, C3221, C3239), lucifer yellow CH (L453, L682, L1177, L12926) and sulforhodamine 101 (S359) in water. |
Other Cascade Blue and Alexa Fluor 405 Derivatives
Cascade Blue acetyl azide (C2284) and Alexa Fluor 405 succinimidyl ester (A30000, A30100) are water-soluble, amine-reactive tracers that can be introduced either by microinjection or by fusion of dye-filled liposomes with cells. Once inside the cell, these derivatives will react with the amine groups of intracellular proteins. Cascade Blue ethylenediamine (C621) and Alexa Fluor 405 cadaverine (previously called Cascade Blue cadaverine, A30675) are aldehyde-fixable fluorophores with reactive properties similar to those of the ethylenediamine derivative of lucifer yellow (A1339). A Cascade Blue dye–labeled 10,000 MW dextran (D1976, Fluorescent and Biotinylated Dextrans—Section 14.5) is also available.
Biocytin and Other Biotin Derivatives
Biocytin (ε-biotinoyl-L-lysine, B1592, ) and biotin ethylenediamine (A1593, ) are microinjectable anterograde and transneuronal tracers. Retrograde transport of biocytin and biotin ethylenediamine in neurons has also been reported. These water-soluble tracers are often used to label neurons during electrophysiological measurements in order to correlate neuronal function with structure and connectivity. Biotin cadaverine (A1594) and biotin-X cadaverine (B1596) have slightly longer spacers than their ethylenediamine counterparts, making the hapten more accessible to the deep biotin-binding site in avidins.
Biocytin, biotin ethylenediamine, biotin cadaverine and biotin-X cadaverine all contain primary amines and can therefore be fixed in cells with formaldehyde or glutaraldehyde and subsequently detected using fluorescent- or enzyme-labeled avidin or streptavidin second-step reagents or with NANOGOLD and Alexa Fluor FluoroNanogold streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). Biocytin hydrazide (B1603) and DSB-X biotin hydrazide (D20653) can serve as aldehyde-fixable tracers and as reactive probes for labeling glycoproteins and nucleic acids (Biotinylation and Haptenylation Reagents—Section 4.2).
As with the reactive lucifer yellow, and Cascade Blue and Alexa Fluor 405 derivatives discussed above, amine- or thiol-reactive biotin derivatives are useful for intracellular labeling applications. The succinimidyl esters of biotin and biotin-X (B1513, B1582) have been used to trace retinal axons in avian embryos. Because they are more water soluble, the sulfosuccinimidyl esters of biotin-X and biotin-XX (B6353, B6352) or the thiol-reactive biocytin maleimide (M1602) may be preferred for these applications.
Fluorescent Biotin Derivatives
Fluorescence of the finer processes of dye-filled neurons may fade rapidly or be obscured by the more intensely stained portions of the neuron, necessitating further amplification of the signal or other ultrastructural detection methods. Lucifer yellow biocytin (L6950), Alexa Fluor 488 biocytin (A12924), Alexa Fluor 546 biocytin (A12923), Alexa Fluor 594 biocytin (A12922), Oregon Green 488 biocytin (O12920) and tetramethylrhodamine biocytin (T12921) each incorporate a fluorophore, biotin and an aldehyde-fixable primary amine into a single molecule, thus enabling researchers to amplify the signals of these tracers with fluorescent or enzyme-labeled avidin or streptavidin conjugates (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6). Although our lucifer yellow cadaverine biotin-X (L2601) lacks a primary amine, it was reported that this tracer was well retained in aldehyde-fixed tissues, even after sectioning, extraction with detergents and several washes. Because fluorescent biocytin derivatives contain free primary amines, they should be even more efficiently fixed by formaldehyde or glutaraldehyde.
Polar fluorescent dyes are commonly used to investigate fusion, lysis and gap-junctional communication and to detect changes in cell or liposome volume. These events are primarily monitored by following changes in the dye's fluorescence caused by interaction with nearby molecules. For example, because the fluorescence of many dyes at high concentrations is quenched, various processes that result in a dilution of the dyes, such as lysis or fusion of fluorescent dye–filled cells or liposomes, can produce an increase in fluorescence, thereby providing an easy method for monitoring these events. Cell–cell and cell–liposome fusion, as well as membrane permeability and transport through gap junctions, can all be monitored using these methods. Furthermore, a fluorogenic substrate such as fluorescein diphosphate (FDP, F2999; Detecting Enzymes That Metabolize Phosphates and Polyphosphates—Section 10.3) can be incorporated within a cell or vesicle that lacks the enzymatic activity to generate a fluorescent product; subsequent fusion with a cell or vesicle that contains the appropriate enzyme will generate a fluorescent product.
An ultrasensitive fusion assay that can be used to follow fusion of single vesicles utilizes an almost nonfluorescent potassium salt of an ion-sensitive indicator such as fluo-3 (F1240, F3715; Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3) in one vesicle and a polyvalent ion such as Ca2+—or perhaps better La3+, which causes greater enhancement of the fluorescence of fluo-3—in a second vesicle.
Fluorescein Derivatives
The self-quenching of fluorescein derivatives provides a means of determining their concentration in dynamic processes such as lysis or fusion of dye-filled cells or liposomes (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). Calcein (C481)—a polyanionic fluorescein derivative that has about six negative and two positive charges at pH 7 ()—as well as BCECF (B1151, ), carboxyfluorescein (C194, C1904), the 5-isomer of Oregon Green 488 carboxylic acid (O6146) and fluorescein-5-(and 6-)sulfonic acid (F1130) are all soluble in water at >100 mM at pH 7. Unlike the other fluorescein derivatives, both calcein and Oregon Green 488 carboxylic acid exhibit fluorescence that is essentially independent of pH between 6.5 and 12.
These green-fluorescent polar tracers are widely used for investigating:
Fluorescence of calcein (but not of carboxyfluorescein or fluorescein sulfonic acid) is strongly quenched by Fe3+, Co2+, Cu2+ and Mn2+ at physiological pH but not by Ca2+ or Mg2+ ions. Monitoring the fluorescence level of cells that have been loaded with calcein (or its AM ester, see below) may provide an easy means for following uptake of Fe3+, Co2+, Cu2+, Mn2+ and certain other metals through ion channels. Increases in the internal volume of lipid vesicles and virus envelopes cause a decrease in Co2+-induced quenching of calcein, a change that can be followed fluorometrically. In addition, the Co2+-quenched calcein complex is useful for both lysis and fusion assays (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). Calcein is a preferred reagent for following volume changes because its fluorescence is not particularly sensitive to either pH or physiological concentrations of other ions.
We prepare a high-purity grade of calcein (C481) that is generally >97% pure by HPLC. The chemical structure assigned to "calcein" in various literature references and by commercial sources has been inconsistent; our structure () has been confirmed by NMR spectroscopy, and we believe that several past assignments of other structures to calcein were incorrect. We also offer a high-purity grade of 5-(and 6-)carboxyfluorescein (C1904) that contains essentially no polar or nonpolar impurities that might alter transfer rates of the dye between vesicles and cells.
Cell-Permeant Fluorescein Derivatives
Cell-permeant versions of carboxyfluorescein, fluorescein sulfonic acid, calcein and the Oregon Green dyes permit passive loading of cells (Viability and Cytotoxicity Assay Reagents—Section 15.2). Acid hydrolysis of nonfluorescent carboxyfluorescein diacetate (CFDA; C195, C1361, C1362; Viability and Cytotoxicity Assay Reagents—Section 15.2) to fluorescent carboxyfluorescein has been used to detect the fusion of dye-loaded clathrin-coated vesicles with lysosomes. CFDA has also been used to investigate cell–cell communication in plant cells. A probenecid-inhibitable anion-transport mechanism permits loading of carboxyfluorescein diacetate and Oregon Green 488 carboxylic acid diacetate (O6151, ; Viability and Cytotoxicity Assay Reagents—Section 15.2) into hyphal tip-cells of some fungi.
Calcein AM (), but not the AM or acetate esters of BCECF or CFDA, is reported to differentially label lymphocytes, permitting their resolution into two populations based on fluorescence intensity, only one of which is taken up by lymphoid organs. This unique property makes calcein AM a useful probe for determining the lymph node homing potential of lymphocytes.
In an important technique for studying gap junctional communication, cells are simultaneously labeled with calcein AM (C1430, C3099, C3100MP) and DiI (D282, D3911, V22885; Tracers for Membrane Labeling—Section 14.4) and then mixed with unlabeled cells (Figure 14.3.5). When gap junctions are established, only the cytosolic calcein tracer (but not the DiI membrane probe) is transferred from the labeled cell to the unlabeled cell. Thus, after gap-junctional transfer, the initially unlabeled cells exhibit the green fluorescence of calcein but not the red fluorescence of DiI. This assay can be followed by either imaging or flow cytometry. In addition, calcein AM and DiI have been combined for use in following cell fusion and for analysis of cholesterol processing by macrophages following ingestion of apoptotic cells.
Figure 14.3.5 A simple technique for the study of gap junctional communication. A population of cells are labeled simultaneously with DiI (D282, D3911) and calcein AM (C1430, C3099, C3100MP), and then mixed with an unlabeled cell population (panel A). The formation of gap junctions allows the cytosolic tracer calcein to cross into the unlabeled cell, while the membrane-bound DiI does not (panel B). Cells from the initial unlabeled population that have taken part in gap junctional communication will therefore display the green fluorescence of calcein while lacking the red-fluorescent signal of DiI.
Fluorescein Substitutes
Fluorophores and Their Amine-Reactive Derivatives—Chapter 1 describes several of our proprietary green-fluorescent dyes that have exceptional optical properties, including our Alexa Fluor 488 dye (Alexa Fluor Dyes Spanning the Visible and Infrared Spectrum—Section 1.3), BODIPY FL dye (BODIPY Dye Series—Section 1.4) and Oregon Green dyes (Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5). Not only do these innovative fluorescein substitutes exhibit high quantum yields in aqueous solution, but the dyes are significantly more photostable than fluorescein (Figure 14.3.6) and their fluorescence is less sensitive to pH (Figure 14.3.7). Their greater photostability makes them the preferred green-fluorescent dyes for fluorescence microscopy. In addition to their membrane-permeant versions, which are described in Viability and Cytotoxicity Assay Reagents—Section 15.2, highly water-soluble derivatives of these fluorescein substitutes are available for use as polar tracers:
- Oregon Green 514 carboxylic acid (O6138, ), which is highly photostable and has little pH sensitivity at near-neutral pH
- BODIPY 492/515 disulfonic acid (D3238), which has narrow spectral bandwidths and bright green, pH-independent fluorescence
- Carboxy-2',7'-dichlorofluorescein (C368), which has a lower pKa than fluorescein
Figure 14.3.6 Comparison of photostability of green-fluorescent antibody conjugates. The following fluorescent goat anti–mouse IgG antibody conjugates were used to detect mouse anti–human IgG antibody labeling of human anti-nuclear antibodies in HEp-2 cells on prefixed test slides (INOVA Diagnostics Corp.): Oregon Green 514 (O6383, ), Alexa Fluor 488 (A11001, ), BODIPY FL (B2752, ), Oregon Green 488 (O6380, ) or fluorescein (F2761, ). Samples were continuously illuminated and viewed on a fluorescence microscope using a fluorescein longpass filter set. Images were acquired every 5 seconds. For each conjugate, three data sets, representing different fields of view, were averaged and then normalized to the same initial fluorescence intensity value to facilitate comparison. |
Figure 14.3.7 Comparison of pH-dependent fluorescence of the Oregon Green 488 (), carboxyfluorescein () and Alexa Fluor 488 () fluorophores. Fluorescence intensities were measured for equal concentrations of the three dyes using excitation/emission at 490/520 nm. |
Sulforhodamine 101 (S359, ) and sulforhodamine B (S1307, ) are orange- to red-fluorescent, very water-soluble sulfonic acid tracers with strong absorption and good photostability. Sulforhodamine 101—the precursor to reactive Texas Red derivatives—has been the preferred red-fluorescent polar tracer for use in combination with lucifer yellow CH, carboxyfluorescein or calcein (). Activity-dependent uptake of sulforhodamine 101 during nerve stimulation has been reported. Sulforhodamine 101 specifically labels astrocytes both in vivo and in acute brain slice preparations. Labeling is accomplished by application of a sulforhodamine 101 solution (1–25 µM in artificial cerebrospinal fluid) to the tissue for 1–5 minutes and is stable for several hours. This technique is particularly useful for cellular-context identification in conjunction with calcium imaging using Oregon Green 488 BAPTA-1, fluo-4 and related fluorescent indicators (Fluorescent Ca2+ Indicators Excited with Visible Light—Section 19.3). Because it is chemically stable, can be prepared in high purity and has a fluorescence quantum yield of nearly 1.0, we have included sulforhodamine 101 in our Reference Dye Sampler Kit (R14782, Fluorescence Microscopy Accessories and Reference Standards—Section 23.1), along with four other dyes whose spectra cover the visible wavelengths.
Sulforhodamine B is an alternative to sulforhodamine 101 for investigating neuronal morphology, preparing fluorescent liposomes, studying cell–cell communications and labeling elastic and collagen fibers.
7-Hydroxycoumarin-3-carboxylic acid (H185) is a blue-fluorescent polar tracer (excitation/emission maxima of ~388/445 nm) with uses that complement those of calcein and the other green-fluorescent polar tracers. The membrane-permeant AM ester of calcein blue (C1429, Viability and Cytotoxicity Assay Reagents—Section 15.2), another coumarin-based tracer, can be used for passive loading of cells.
Polysulfonated Pyrenes
HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid, also known as pyranine, H348; ) is a unique pH-sensitive tracer. It fluoresces blue in acidic solutions and in acidic organelles, but fluoresces green in more basic organelles. In addition to its use as a probe for proton translocation, HPTS has been employed for intracellular labeling of neurons and as a fluid-phase endocytic tracer in catecholamine-secreting PC12 rat pheochromocytoma cells. HPTS forms a nonfluorescent complex with the cationic quencher DPX (X1525), and several assays have been described that monitor the increase in HPTS fluorescence that occurs upon lysis or fusion of liposomes or cells containing this quenched complex (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). HPTS has also been used as a viscosity probe in unilamellar phospholipid vesicles.
The pH-insensitive 8-aminopyrene-1,3,6-trisulfonic acid (APTS, A6257) and 1,3,6,8-pyrenetetrasulfonic acid (P349) are extremely soluble in water (>25%); they have been utilized as blue-fluorescent tracers. As with HPTS, the fluorescence of APTS and 1,3,6,8-pyrenetetrasulfonic acid is quenched by DPX (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3) and by the cationic spin label CAT 1 (T506, see below). These quenched-fluorophore complexes are useful for following lysis of cells and liposomes.
The polyanionic dye ANTS (A350) is often used in combination with the cationic quencher DPX (X1525) for membrane fusion or permeability assays, including complement-mediated immune lysis (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). Thallium (Tl+) and cesium (Cs+) ions quench the fluorescence of ANTS, pyrenetetrasulfonic acid and some other polyanionic fluorophores. A review by Garcia describes how this quenching effect can be utilized to determine transmembrane ion permeability. The unusually high Stokes shift of ANTS in water (>150 nm) separates its emission from much of the autofluorescence of biological samples. An approximately fourfold enhancement of the quantum yield of ANTS is induced by D2O—a spectral characteristic that has been used to determine water permeability in red blood cell ghosts and kidney collecting tubules. ANTS has also been employed as a neuronal tracer.
Lanthanide Chelates
Terbium ion (Tb3+ from TbCl3, T1247) forms a chelate with dipicolinic acid (DPA) that is ~10,000 times more fluorescent than free Tb3+. Fusion of vesicles that have been separately loaded with DPA and Tb3+ results in enhanced fluorescence, providing the basis for liposome fusion assays (Assays of Volume Change, Membrane Fusion and Membrane Permeability—Note 14.3). The fluorescence emission spectrum of the Tb3+/DPA complex exhibits two sharp spectral peaks at 491 nm and 545 nm and a lifetime of several milliseconds. Because DPA is a major constituent of bacterial spores, Tb3+/DPA complex luminescence provides a straightforward yet sensitive method for their detection.
TOTO, YOYO and SYTO Nucleic Acid Stains
Our high-affinity nucleic acid stains, including TOTO-1 and YOYO-1 (T3600, Y3601; Nucleic Acid Detection and Genomics Technology—Chapter 8), form tight complexes with nucleic acids with slow off-rates for release of the dye. Consequently, nucleic acids that have been prelabeled with these dyes can be traced in cells following microinjection or during gene transfer (). The cell-to-cell transport via plasmodesmata of TOTO-1 dye–labeled RNA, single-stranded DNA and double-stranded DNA has been determined following microinjection of the labeled nucleic acids into plant cells.
Khoobehi and Peyman have demonstrated the use of our cell-permeant SYTO nucleic acid stains as ophthalmological tracers of blood flow. White blood cells were passively loaded with the green-fluorescent SYTO 16 dye (S7578, Nucleic Acid Stains—Section 8.1) or the red-fluorescent SYTO 59 dye (S11341, Nucleic Acid Stains—Section 8.1). Red blood cell membranes were labeled with DiD (D307, Tracers for Membrane Labeling—Section 14.4). By using an argon-ion laser to excite SYTO 16 and a red light–emitting He-Ne laser to excite both SYTO 59 and DiD, they were able to follow the relative mobility of the two types of blood cells.
UV photolysis of photoactivatable fluorescent dyes provides a means of controlling—both spatially and temporally—the release of fluorescent tracers (Photoactivatable Reagents, Including Photoreactive Crosslinkers and Caged Probes—Section 5.3). Thus, caged dyes enable researchers to follow the movement of individual molecules and cellular structures, as well as to study cell lineage in live organisms (). CMNB-caged fluorescein (F7103, ) is colorless and nonfluorescent until it is photolyzed at <365 nm to the intensely green-fluorescent free dye. Movement of the liberated fluorophore from the site of photolysis can then be followed. The succinimidyl ester of CMNB-caged carboxyfluorescein (C20050, Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5), which is water soluble at pH 7, is useful for preparation of other caged dye tracers, including those of proteins.
Anterograde and retrograde tracing of neurons has utilized a wide variety of fluorescent and nonfluorescent probes (Figure 14.3.8). Among the retrograde and anterograde tracers are biotin derivatives and polar fluorescent dyes (described in this section), lipophilic tracers such as DiI (D282, Tracers for Membrane Labeling—Section 14.4), dextran conjugates (Fluorescent and Biotinylated Dextrans—Section 14.5), fluorescent microspheres (Microspheres and Qdot Nanocrystals for Tracing—Section 14.6) and protein conjugates, including lectins (Protein Conjugates—Section 14.7).
Figure 14.3.8 Structural schematic of a neuron indicating the directions of retrograde and anterograde transport.
True Blue and Nuclear Yellow
The popular retrograde tracer true blue (T1323) is a UV light–excitable, divalent cationic dye that stains the cytoplasm with blue fluorescence. For two-color neuronal mapping, true blue has been combined with longer-wavelength tracers such as nuclear yellow (Hoechst S769121, N21485) or diamidino yellow, which primarily stain the neuronal nucleus with yellow fluorescence (, , ). Fluorescent microspheres have also been used as a counterstain with true blue. True blue is reported to be a less cytotoxic retrograde tracer than Fluoro-Gold and to be a more efficient retrograde tracer than diamidino yellow. Both true blue and nuclear yellow are stable when subjected to immunohistochemical processing and can be used to photoconvert DAB into an insoluble, electron-dense reaction product.
Hydroxystilbamidine and Aminostilbamidine
Hydroxystilbamidine methanesulfonate (H22845, ) was originally developed as a trypanocide and has been identified by Wessendorf as the active component of a dye that was named Fluoro-Gold by Schmued and Fallon and later sold for retrograde tracing under that trademark by Fluorochrome, Inc. The use of hydroxystilbamidine as a histochemical stain and as a retrograde tracer for neurons apparently goes back to the work of Snapper and collaborators in the early 1950s who, while studying the effects of hydroxystilbamidine on multiple myeloma, showed that therapeutically administered hydroxystilbamidine gives selective staining of ganglion cells. The comprehensive article by Wessendorf also describes several other early applications of hydroxystilbamidine that do not include its therapeutic uses:
- As an AT-selective, nonintercalating nucleic acid stain for discriminating between DNA and RNA (Nucleic Acid Stains—Section 8.1)
- For histochemical staining of DNA, mucosubstances and elastic fibers, as well as mast cells
- As a ribonuclease inhibitor
- For lysosomal staining of live cells (Probes for Lysosomes, Peroxisomes and Yeast Vacuoles—Section 12.3)
The weakly basic properties of hydroxystilbamidine that result in its uptake by lysosomes are reportedly important for its mechanism of retrograde transport. We developed a product that we called hydroxystilbamidine (formerly catalog number H7599) in April 1995; however, we subsequently discovered that the chemical structure of our original "hydroxystilbamidine" corresponded to a novel dye that we now call aminostilbamidine (A22850, ). Apparently, aminostilbamidine functions at least as well as authentic hydroxystilbamidine as a tracer. Aminostilbamidine, however, does not show the spectral shifts with DNA and RNA that are observed with authentic hydroxystilbamidine.
Propidium Iodide and DAPI for Retrograde Tracing
A variety of other low molecular weight dyes have been used as fluorescent retrograde neuronal tracers. These include propidium iodide (P1304MP, P21493) and DAPI. Both propidium iodide and DAPI can be used to photoconvert DAB into an insoluble, electron-dense product. The lactate salt of DAPI (D3571) has much higher water solubility than the chloride salt (D1306, D21490), making it the preferred form for microinjection.
The Nissl substance, described by Franz Nissl more than 100 years ago, is unique to neuronal cells. Composed of an extraordinary amount of rough endoplasmic reticulum, the Nissl substance reflects the unusually high protein synthesis capacity of neuronal cells. Various fluorescent or chromophoric "Nissl stains" have been used for this counterstaining, including acridine orange, ethidium bromide, neutral red (N3246, Viability and Cytotoxicity Assay Reagents—Section 15.2), cresyl violet, methylene blue, safranin-O and toluidine blue-O. We have developed five fluorescent Nissl stains (Fluorescence characteristics of NeuroTrace fluorescent Nissl stains—Table 14.2) that not only provide a wide spectrum of fluorescent colors for staining neurons, but also are far more sensitive than the conventional dyes:
- NeuroTrace 435/455 blue-fluorescent Nissl stain (N21479, )
- NeuroTrace 500/525 green-fluorescent Nissl stain (N21480; , , )
- NeuroTrace 515/535 yellow-fluorescent Nissl stain (N21481, )
- NeuroTrace 530/615 red-fluorescent Nissl stain (N21482; , )
- NeuroTrace 640/660 deep red–fluorescent Nissl stain (N21483)
In addition, the Nissl substance redistributes within the cell body in injured or regenerating neurons. Therefore, these Nissl stains can also act as markers for physically or chemically induced neurostructural damage. Staining by the Nissl stains is completely eliminated by pretreatment of tissue specimens with RNase; however, these dyes are not specific stains for RNA in solutions. The strong fluorescence (emission maximum ~515–520 nm) of NeuroTrace 500/525 green-fluorescent Nissl stain (N21480) makes it the preferred dye for use as a counterstain in combination with orange- or red-fluorescent neuroanatomical tracers such as DiI (D282, D3911, V22885; Tracers for Membrane Labeling—Section 14.4; ).
The highly water-soluble cationic spin label 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-1-oxyl iodide (CAT 1, T506) has been used to:
Polar tracers such as Cascade Blue hydrazide, lucifer yellow CH, Alexa Fluor 488 hydrazide and biocytin penetrate even the finest structures of neurons and other cells; however, as the thickness of the sample is decreased, the fluorescence signal is reduced. Consequently, it may be necessary to further amplify the signal by secondary detection methods.
We provide rabbit polyclonal antibodies to the Alexa Fluor 488, Alexa Fluor 405/Cascade Blue, lucifer yellow, fluorescein, BODIPY FL, tetramethylrhodamine and Texas Red fluorophores (Anti-Dye and Anti-Hapten Antibodies—Section 7.4, Anti-fluorophore and anti-hapten antibodies—Table 7.8) and a vast number of dye- or enzyme-labeled anti-rabbit antibodies (Secondary Immunoreagents—Section 7.2, Summary of Molecular Probes secondary antibody conjugates—Table 7.1). Our polyclonal and monoclonal antibodies to fluorescein crossreact strongly with the Oregon Green dyes and somewhat with Rhodamine Green fluorophores, and our anti-tetramethylrhodamine and anti–Texas Red antibodies crossreact with tetramethylrhodamine, Lissamine rhodamine B, Rhodamine Red and Texas Red dyes.
Our Tyramide Signal Amplification (TSA) Kits (TSA and Other Peroxidase-Based Signal Amplification Technology—Section 6.2, Tyramide Signal Amplification (TSA) Kits—Table 6.1) can also be utilized to detect aldehyde-fixed biotin derivatives or, in combination with antibodies to aldehyde-fixed fluorophore-labeled polar tracers, to further amplify the signal. Following fixation, biotinylated tracers such as biocytin and biotin ethylenediamine, can be detected using the reagents in our TSA Kits that contain horseradish peroxidase streptavidin and a fluorescent tyramide.
The relatively small size and easy penetration into tissues makes the streptavidin conjugates of the NANOGOLD, Alexa Fluor 488 FluoroNanogold and Alexa Fluor 594 FluoroNanogold 1.4 nm gold clusters (N24918, A24926, A24927; Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) useful for ultrastructural studies of biotinylated tracers, particularly in combination with the LI Silver (LIS) Enhancement Kit (L24919, Secondary Immunoreagents—Section 7.2). Biotinylated polar tracers can also be detected with light microscopy using the Diaminobenzidine (DAB) Histochemistry Kit #3 (D22187, TSA and Other Peroxidase-Based Signal Amplification Techniques—Section 6.2).
Our Influx pinocytic cell-loading reagent (I14402) works via a rapid and simple technique based on the osmotic lysis of pinocytic vesicles, an approach introduced by Okada and Rechsteiner. The probe is simply mixed at high concentration with the Influx reagent blended into growth medium, then incubated with live cells to allow pinocytic uptake of the surrounding solution. Subsequent transfer of the cells to a slightly hypotonic medium results in bursting of the pinocytic vesicles within the cells and the release of the probe into the cytosol (Figure 14.3.9, , ).
The Influx pinocytic cell-loading reagent is highly effective for loading a diverse array of probes—including calcein (), Alexa Fluor hydrazides (), dextran conjugates of fluorophores and ion indicators (), fura-2 salts, Oregon Green 514 dye–labeled tubulin, Alexa Fluor 488 dye–labeled actin, heparin, hydroxyurea, DNA, oligonucleotides, and Qdot nanocrystals —into a variety of cell lines. We (or other researchers) have successfully tested the reagent and loading method with:
- Bovine pulmonary artery endothelial cells (BPAEC)
- Human epidermoid carcinoma cells (A431)
- Human T-cell leukemia cells (Jurkat)
- Murine fibroblasts (NIH 3T3 and CRE BAG 2)
- Murine monocyte-macrophages (RAW264.7 and J774A.1)
- Murine myeloma cells (P3x63AG8)
- Rat basophilic leukemia cells (RBL)
More than 80% of the cells remained viable, as determined by subsequent exclusion of propidium iodide.
In addition to the Influx pinocytic cell-loading reagent and cell growth medium, all that is required to perform the loading procedure is sterile deionized water and the fluorescent probe or other polar molecule of interest. Cell labeling can be accomplished in a single 30-minute loading cycle and may be enhanced by repetitive loading. Although most types of cells load quickly and easily, optimal conditions for loading must be determined for each cell type. It is also important to note that cell-to-cell variability in the degree of loading is typical () and that higher variability is generally observed when using large compounds, such as >10,000 MW dextrans and proteins.
The Influx pinocytic cell-loading reagent is packaged as a set of 10 tubes (I14402), each containing sufficient material to load 50 samples of cells grown on coverslips following the protocol provided (Influx Pinocytic Cell-Loading Reagent). Cells in suspension or in culture flasks may also be easily loaded; however, the number of possible cell loadings will depend on the cell suspension volume or size of culture flask used. The information provided with the Influx reagent includes general guidelines and detailed suggestions for optimizing cell loading. Use of the custom coverslip mini-rack or coverslip maxi-rack (C14784, C24784; Fluorescence Microscopy Accessories and Reference Standards—Section 23.1) facilitates cell loading and slide handling when using the Influx reagent.
Figure 14.3.9 Principle of the Influx reagent pinocytic cell-loading method (I14402). Cultured cells are placed in hypertonic Influx loading reagent (panel A), along with the material to be loaded into the cells (yellow fluid, panel B), allowing the material to be carried into the cells via pinocytic vesicles. When the cells are placed in hypotonic medium, the pinocytic vesicles burst (panel C), releasing their contents into the cytosol (panel D).P2X7 receptor–expressing cells such as macrophages and thymocytes exhibit reversible pore opening that can be exploited to provide an entry pathway for intracellular loading of both cationic and anionic fluorescent dyes with molecular weights of up to 900 daltons. Pore opening is induced by treatment with 5 mM ATP for five minutes and subsequently reversed by addition of divalent cations (Ca2+ or Mg2+). Dyes that have been successfully loaded into macrophage cells by this method include:
- Ca2+ indicator: fura-2 (F1200, F6799; Fluorescent Ca2+ Indicators Excited with UV Light—Section 19.2)
- pH indicator: HPTS (H348)
- Aqueous tracers: lucifer yellow CH (L453, L682, L1177, L12926) and 6-carboxyfluorescein (C1360, Fluorescein, Oregon Green and Rhodamine Green Dyes—Section 1.5)
- Nucleic acid stain: YO-PRO-1 (Y3603, Nucleic Acid Stains—Section 8.1)
One of the most potent and widely used P2X receptor agonists, BzBzATP (2'-(or 3'-)O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, B22358; Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3), is available. BzBzATP has more general applications for site-directed irreversible modification of nucleotide-binding proteins via photoaffinity labeling; see Probes for Protein Kinases, Protein Phosphatases and Nucleotide-Binding Proteins—Section 17.3 for more information on our nucleotide analogs.
Cat # | Links | MW | Storage | Soluble | Abs | EC | Em | Solvent | Notes |
A1593 | 367.30 | NC | DMF, DMSO | <300 | none | ||||
A1594 | 442.50 | NC | DMF, DMSO | <300 | none | ||||
A6257 | 523.39 | D,L | H2O | 424 | 19,000 | 505 | pH 7 | ||
A10436 | 570.48 | D,L | H2O | 493 | 71,000 | 517 | pH 7 | ||
A10437 | 730.74 | D,L | H2O | 576 | 86,000 | 599 | pH 7 | 1 | |
A10438 | 758.79 | D,L | H2O | 588 | 97,000 | 613 | pH 7 | 1 | |
A10439 | 349.29 | L | H2O, DMSO | 345 | 13,000 | 445 | pH 7 | ||
A10441 | 730.74 | FF,L | H2O | 576 | 86,000 | 599 | pH 7 | 2 | |
A10442 | 758.79 | FF,L | H2O | 588 | 97,000 | 613 | pH 7 | 2 | |
A12922 | 1141.31 | D,L | DMSO, H2O | 591 | 80,000 | 618 | pH 7 | ||
A12923 | 1209.66 | D,L | DMSO, H2O | 556 | 99,000 | 572 | pH 7 | ||
A12924 | 974.98 | D,L | DMSO, H2O | 494 | 62,000 | 520 | pH 7 | ||
A20501MP | ~1150 | D,L | H2O | 554 | 150,000 | 567 | pH 7 | ||
A20502 | ~1200 | D,L | H2O | 649 | 250,000 | 666 | pH 7 | ||
A22850 | 471.55 | F,D,L | H2O, DMSO | 361 | 17,000 | 536 | pH 7 | ||
A30000 | 1028.26 | F,DD,L | H2O, DMSO | 400 | 35,000 | 424 | pH 7 | 3, 4, 5 | |
A30627 | 584.52 | F,D,L | H2O, DMSO | 353 | 20,000 | 437 | MeOH | 6 | |
A30629 | 895.07 | F,D,L | H2O, DMSO | 494 | 77,000 | 518 | pH 7 | 6, 7, 8 | |
A30632 | ~1220 | F,D,L | H2O, DMSO | 651 | 250,000 | 672 | MeOH | 6 | |
A30634 | ~950 | D,L | H2O, DMSO | 624 | 110,000 | 643 | pH 7 | ||
B1151 | 520.45 | L | pH >6 | 503 | 90,000 | 528 | pH 9 | 9 | |
B1370 | 831.01 | L | DMF, pH >6 | 494 | 75,000 | 518 | pH 9 | 9 | |
B1513 | 341.38 | F,D | DMF, DMSO | <300 | none | ||||
B1582 | 454.54 | F,D | DMF, DMSO | <300 | none | ||||
B1592 | 372.48 | NC | H2O | <300 | none | ||||
B1596 | 555.65 | NC | DMF, DMSO | <300 | none | ||||
B1603 | 386.51 | D | pH >6, DMF | <300 | none | ||||
B6352 | 669.74 | F,D | DMF, pH >6 | <300 | none | 4 | |||
B6353 | 556.58 | F,D | DMF, pH >6 | <300 | none | 4 | |||
B10570 | 644.70 | L | DMSO | 494 | 68,000 | 523 | pH 9 | 9 | |
C194 | 376.32 | L | pH >6, DMF | 492 | 75,000 | 517 | pH 9 | 9 | |
C368 | 445.21 | L | pH >6, DMF | 504 | 107,000 | 529 | pH 8 | 10 | |
C481 | 622.54 | L | pH >5 | 494 | 77,000 | 517 | pH 9 | 11, 12 | |
C1430 | 994.87 | F,D | DMSO | <300 | none | 14 | |||
C1904 | 376.32 | L | pH >6, DMF | 492 | 78,000 | 517 | pH 9 | 9, 15 | |
C2284 | 607.42 | F,D,LL | H2O, MeOH | 396 | 29,000 | 410 | MeOH | 3, 16 | |
C3099 | 994.87 | F,D | DMSO | <300 | none | 2, 14 | |||
C3100MP | 994.87 | F,D | DMSO | <300 | none | 14 | |||
D1306 | 350.25 | L | H2O, DMF | 342 | 28,000 | 450 | pH 7 | ||
D3571 | 457.49 | L | H2O, MeOH | 342 | 28,000 | 450 | pH 7 | ||
D20653 | 341.45 | D | DMSO | <300 | none | 18 | |||
D21490 | 350.25 | L | H2O, DMF | 342 | 28,000 | 450 | pH 7 | 15 | |
F1130 | 478.32 | D,L | H2O, DMF | 495 | 76,000 | 519 | pH 9 | 9 | |
F7103 | 826.81 | FF,D,LL | H2O, DMSO | 333 | 15,000 | none | DMSO | 16, 19, 20 | |
H185 | 206.15 | L | pH >6, DMF | 386 | 29,000 | 448 | pH 10 | 21 | |
H348 | 524.37 | D,L | H2O | 454 | 24,000 | 511 | pH 9 | 22 | |
H22845 | 472.53 | F,D,L | H2O, DMSO | 345 | 31,000 | 450 | pH 5 | 23 | |
L1338 | 659.51 | F,D,L | H2O | 426 | 11,000 | 531 | pH 7 | 26 | |
L6950 | 850.03 | D,L | H2O | 428 | 11,000 | 532 | pH 7 | ||
M395 | 538.40 | L | H2O | 404 | 29,000 | 435 | pH 8 | 27 | |
M1602 | 523.60 | F,D | pH >6, DMF | <300 | none | ||||
N21479 | see Notes | F,D,L | DMSO | 435 | see Notes | 457 | H2O/RNA | 2, 28, 29 | |
N21480 | see Notes | F,D,L | DMSO | 497 | see Notes | 524 | H2O/RNA | 2, 28, 29 | |
N21481 | see Notes | F,D,L | DMSO | 515 | see Notes | 535 | H2O/RNA | 2, 28, 29 | |
N21482 | see Notes | F,D,L | DMSO | 530 | see Notes | 619 | H2O/RNA | 2, 28, 29 | |
N21483 | see Notes | F,D,L | DMSO | 644 | see Notes | 663 | H2O/RNA | 2, 28, 29 | |
O6138 | 512.36 | L | pH >6, DMF | 506 | 86,000 | 526 | pH 9 | 30 | |
O6146 | 412.30 | L | pH >6, DMF | 492 | 85,000 | 518 | pH 9 | 31 | |
O12920 | 887.39 | L | DMSO, H2O | 495 | 66,000 | 522 | pH 9 | 31 | |
S1129 | 518.43 | F,D | DMSO | <300 | none | 32 | |||
T506 | 341.25 | F,D | H2O, MeOH | <300 | none | ||||
T12921 | 869.09 | D,L | DMSO | 554 | 103,000 | 581 | pH 7 | ||
X1525 | 422.16 | D | H2O | 259 | 8800 | none | H2O | ||
For Research Use Only. Not for use in diagnostic procedures.


text:
Company Profile : "Intergold (India) Pvt Ltd" Belongs to GEMS & JEWELLERY INDUSTRY. We are the Leaders in Diamond-studded, Gold & Platinum Jewellery. We hold a large share of Market, on the International level. The company will retain its leadership position in the market and capture even more greater share in the International Scenario.
Currently No Jobs has been posted by this Recruiter. |


text:
Rookie Scotty McKnight Tweets his Thoughts on Camp
Scotty McKnight talks to fans on twitter about signing footballs, what it's been like so far for the rookie and what he's doing in the evening.
Read MoreMother Nature is a Jets Fan
Good morning tweeps! Beautiful morning out here in Jersey...mother nature must be happy...
Read MoreRookie WR Scotty McKnight to Wear #15
Got settled in at the facility today. Excited to be in the building and start gettin to...
Read More


text:
Browse by top tags:
Ryan Dittmar: Ski tester, backcountry ripper, uses explosives for a living Copyright: Liam Doran
Megan Brown Brent: Two-time NCAA All-American, 2000 Skiercross World Champ, ski model Copyright: Liam Doran
Jay Bristow: COO of MND Group, which sells equipment for mountain development and safety; Ruby Mountain heliski guide Copyright: Liam Doran
Sam Flickinger: Former Editor of Ski Racing Magazine, current technical writer Copyright: Liam Doran
Sharelle Rodman: Former racer, seasoned ski tester, Park City realtor and local ripper Copyright: Liam Doran
Dan Kasper: OnTheSnow Senior Managing Editor and longtime big mountain ripper Copyright: Liam Doran
Susi Muecke: Grew up skiing in Germany and the Swiss Alps, PSIA Level II instructor at Snowbird Copyright: Liam Doran
Patrick Crawford: Former OnTheSnow Global Content Director, co-founder for Freeskier Magazine Copyright: Liam Doran
Sue Minecci: Former NCAA Division I racer, Alta ripper Copyright: Liam Doran
Bret Zawacki: Former college racer, Snowbird ski instructor of 33 years
Caroline Gleich: Snowbird/Alta local, Patagonia ambassador, Warren Miller athlete Copyright: Liam Doran
Josef Stoeger: PSIA examiner and trainer, Snowbird instructor Copyright: Liam Doran
Georgia Dumais: PSIA Level III Alpine and Telemark instructor at Snowbird Copyright: Liam Doran
Ben Attridge: Deer Valley ski instructor and local ripper Copyright: Liam Doran
Jody Curtis: Former ski racer, ski tester and Utah ripper Copyright: Liam Doran
KC Gaudet: Former college racer, big mountain skiing competitor, Alta ripper Copyright: Liam Doran
Krista Crabtree: Former ski racer, former SKI Magazine women's ski test director and current OnTheSnow ski test director Copyright: Liam Doran
Steven Bills: Former Director of Snowbird Mountain School, lifetime member of PSIA Copyright: Liam Doran
Nona Weatherbee: PSIA Level III Alpine instructor, has taught at the 'Bird since 1971 Copyright: Liam Doran
Donny O'Neill: Former OnTheSnow Assistant Editor, current Assistant Editor at Freeskier Magazine Copyright: Liam Doran
Erik Schlopy: Three-time Olympian and seven-time National Alpine skiing Champ, super dad Copyright: Liam Doran
Tim Witman: Former NCAA Division I ski racer, super gear tester, winner of most skis tested over three days at the 'Bird Copyright: Liam Doran
Resort: Snowbird
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text:
WASHINGTON, D.C. (CNET) – Internet tax supporters are hoping that a vote in the U.S. Senate as early as today will finally give them enough political leverage to require Americans to pay sales taxes when shopping online.
Sens. Mike Enzi (R-Wy.) and Dick Durbin (D-Ill.) are expected to offer an amendment to a Democratic budget resolution this week that, by allowing states to "collect taxes on remote sales," is intended to usher in the first national Internet sales tax.
"We're working overtime in pushing this, talking to our members, activating our grassroots," says Stephen Schatz, a spokesman for the National Retail Federation. The group's board members include OfficeMax, Macy's, the Container Store, and Saks, which argue it's only fair to force Americans to pay sales taxes when buying from online retailers.
The justification for the proposal reprises arguments that state tax collectors have made for at least a decade: online retailers that don't always collect taxes are unreasonably depriving state governments of revenue and enjoy an unfair competitive advantage over big box retailers that do collect taxes. On the other hand, there are close to 10,000 jurisdictions that can levy taxes, each with its own rules and ability to conduct audits, and complying with all of those as a small retailer is not a trivial task.
Taxpayer advocates say an endorsement of a multi-billion dollar tax hike on Americans shouldn't be snuck into an unrelated budget bill (PDF) that's expected to be voted on before senators leave for an Easter recess. The National Taxpayers Union set up a petition to Congress this week calling the amendment "really just a way to unleash state tax collectors on the Internet," and 15 conservative groups sent a letter last week to members of Congress saying an Internet tax law is "is bad news for conservative principles and the cause of limited government."
They're joined by by eBay, an association of small Internet sellers called W R HERE, and NetChoice, which includes Facebook, Yahoo, LivingSocial, and AOL as members.
The proposed amendment "does nothing to address what the Supreme Court says was an unreasonable burden on interstate commerce," says Steve DelBianco, executive director of NetChoice. "It's an unprecedented expansion of state sales tax authority."
Enzi and Durbin sponsored the separate Marketplace Fairness Act of 2013 (S.336), introduced last month, which has 25 other Senate supporters and would authorize state governments to collect taxes from remote sellers with more than $1 million in gross receipts.
But it takes effect only if state governments sufficiently simplify their notoriously labyrinthine tax laws. In New Jersey, for instance, bottled water and cookies are exempt from sales tax (PDF), but bottled soda and candy are taxable. In Rhode Island, buying a mink handbag is taxed, but a mink fur coat is not.
The Enzi-Durbin amendment takes a similar approach, but lacks mandatory simplification and is non-binding. It appears to be intended as a clever political hack: secure plenty of votes on a non-binding Internet tax amendment, then use those vote totals to argue there's sufficient support for S.336 when it's up for a binding vote later.
"The strategy of the bill's supporters is to offer this general amendment and then claim that all the senators that vote for it support the bill," says Brian Bieron, eBay's senior director for federal government relations and global public policy. "That is not just a stretch, it is not accurate. But the game plan is to rack up a sizable vote and then make the claim the bill itself should jump over the Finance Committee and go right to the floor." (Sen. Max Baucus, D-Mont., head of the Senate Finance committee who represents a state with no sales tax, has been less than enthusiastic about S.336.)
A spokesman for Enzi did not respond to queries from CNET yesterday afternoon. Durbin haspreviously said that enacting the Internet tax legislation is "a matter of basic economic fairness."
Enzi's allies, including big box retailers and Sens. Dianne Feinstein (D-Calif.), Al Franken (D-Minn.), Lamar Alexander (R-Tenn.), and Heidi Heitkamp (D-S.D.), hope that they can convince 60 senators to vote for the amendment — a key supermajority that would ease the path for enacting S.336 in the future.
A letter that the Retail Industry Leaders Association sent to all senators on Tuesday says the Enzi-Durbin amendment "simply provides states with the authority — if they so choose — to require online sellers to collect sales taxes just like brick and mortar and many of their dot.com Web sites already do today." RILA's board members include representatives of Best Buy, Home Depot, Walmart, Whole Foods, Target, and AutoZone.
The current legal and political landscape was shaped by a 1992 case called Quill v. North Dakota, in which the U.S. Supreme Court ruled that out-of-state retailers generally don't have to collect sales taxes.
One exception to that rule is a legal concept called "nexus," which means a company can be forced to collect sales taxes if it has a sufficient business presence, which is why Amazon.com wasn't required to collect sales taxes in California until recently. Another exception is the sale of cigarettes, which is covered by the Jenkins Act.
As a practical matter, many Americans already pay sales taxes on Internet purchases, either from big box retailers' Web sites or from online retailers like Amazon.com that have generated nexus by opening warehouses or subsidiaries in more states. Smaller retailers, including Newegg.com, Overstock.com, Blue Nile, Systemax's TigerDirect.com, and eBay sellers are less likely to have nexus, meaning their purchases would arrive tax-free (use taxes, however, may still apply).
Supporters of Internet tax legislation say they're optimistic. "It's bound to happen," says Schatz, from the National Retail Federation. "It's just a matter of when."
(TM and © 2013 CBS Interactive, Inc. and CNET, a CBS-owned company. All Rights Reserved. This material may not be published, broadcast, rewritten, or redistributed.)


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HP unveils slick, iMac-like all-in-one PC Drawing inspiration from Apple's designs is a time-honored tradition throughout the industry—Apple v. Samsung tomfoolery notwithstanding. HP offered an excellent reminder of that this morning... Type: News story Tags: Systems |
56 |
Zotac updates Zbox Nano with Brazos 2.0 We loved the Zotac Zbox Nano AD10 when we reviewed it last year. Into a chassis small enough to fit in the palm of... Type: News story Tags: CPUs Graphics Chipsets Systems |
45 |
Mad Catz keyboard has modular layout, integrated touchscreen There's a new high-end gaming keyboard in town, and it's different to say the least. At the Gamescom expo in Cologne, Germany, Mad Catz... Type: News story Tags: Input devices |
29 |
Razer unveils adjustable mouse with 8200 DPI sensor Well, Razer definitely isn't messing around. The company's new Ouroboros wireless mouse carries a $129.99 price tag, features an 8200-DPI sensor, and is both... Type: News story Tags: Input devices |
41 |
Honey, Zotac shrunk the nettop Zotac's Zbox Nano AD10 is one of the smallest nettops we've ever seen. It's a fantastic mini PC, and now there's new version... Type: News story Tags: Systems |
59 |
Sapphire's Edge HD³ nettop has AMD inside Last June, Sapphire showed us the Edge, a slim little nettop powered by Intel's Atom D510 processor and Nvidia's Ion discrete GPU. The firm... Type: News story Tags: Systems |
17 |
Razer brings Switchblade UI to standalone keyboard Razer was keen to show off its Blade gaming laptop at the Consumer Electronics Show this year, but the system is a little large—and... Type: News story Tags: Input devices |
16 |
Sandy Bridge upgrades come to Zotac mini PCs While we were most excited to see 7-series Mini-ITX boards at Zotac's CES showcase last week, the company also had a number of interesting... Type: News story Tags: Systems |
12 |
Thermaltake brings rubber domes back in style Mechanical keyboards have seen something of a renaissance in recent years—especially in hardcore gaming circles, where keyboards based on Cherry's MX black key switches are... Type: News story Tags: Input devices |
57 |
Cherry MX switches take root in Corsair gaming keyboards IDF — Corsair rolled out a slew of new gaming peripherals at the Intel Developer Forum this week. We were at the big reveal,... Type: News story Tags: Input devices |
59 |
Ultra-slim Ultrabooks strut down the catwalk IDF — Mooly Eden laid out the basic parameters for the new wave of laptops that Intel has been pushing during his IDF keynote... Type: News story Tags: CPUs Systems Mobile computing |
75 |
Sapphire shows A75 mobo, passive Radeons Computex — While visiting Sapphire in Taipei this week, we got a quick look at new motherboards based on AMD's 990FX and upcoming A75 chipsets.... Type: News story Tags: Graphics Motherboards Systems |
31 |
Brazos injected into Zotac barebones nettop With barebones nettops systems built around Ion and CULV underpinnings, it was only a matter of time before Zotac put Brazos inside a... Type: News story Tags: CPUs Graphics Systems |
25 |
$2,899 hand-built PC has unique chassis Announcements about garish, LED-covered "enthusiast" cases make it into my inbox on a weekly basis. Genuinely tasteful and visually appealing cases, on the other hand,... Type: News story Tags: Cases Systems |
46 |
Thermaltake shows mechanical gaming keyboards COMPUTEX — Keyboards with clicky, mechanical keyswitches have gathered a cult following in recent years, and Thermaltake is planning to capitalize on that. Sitting next... Type: News story Tags: Input devices |
52 |
Motion control comes to the PS3 with Sony's PlayStation Move Sony has officially unveiled its motion controller for the PlayStation 3 game console. The wand-style PlayStation Move made its debut at the Game... Type: News story Tags: Input devices Game consoles |
50 |
Zotac reveals Zbox nettop with next-gen Ion Now that Nvidia has officially pulled the curtain off its new Ion, we can tell you about more of the systems based on the... Type: News story Tags: Systems |
15 |
18-button mouse loses OpenOffice label, gains laser Remember the OpenOffice.org mouse we told you about last month? Well, it's gotten a new name, a black paint job, and an adjustable... Type: News story Tags: Input devices |
27 |
Razer Blade Pro swims in the deep end of Kaby Lake | 2 |


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First Defense Immune Boost, 4 fl oz, From Childlife
See all products in:
Vitamin C for Kids
Sinus Formulas
Customer Reviews:
Stock #:
Manufacturer Part #:
In Stock - Usually Ships Within 24 - 48 Hours
We Ship To:
Shipping Rates
Ground Shipping
Order Under $100:$6.99
Order over $100:FREE SHIPPING
Health Tip For stimulating natural immune responses and defending the body. Use daily at the recommended dosages. Use with ChildLife's Echinacea and ChildLife's Vitamin C for extra immune support. First Defense Immune Boost 4 fl oz
Immune support formulas
to be given when needed, to stimulate natural immune responses and defend the body. First Defense Immune Boost 4 fl oz these formulas can also be used periodically for increasing immune support and strength. First Defense Immune Boost 4 fl oz About ChildLife Essentials:
ChildLife offers doctor-developed nutritional formulas you can trust, to help provide your infant or child with the best possible start in life. First Defense Immune Boost 4 fl oz
Along with a healthful diet and careful nurturing, the ChildLife nutritional formulas will support your child through their formative years and help create a solid foundation for good health throughout life.Make ChildLife nutritional supplements a regular part of your child's daily routine. First Defense Immune Boost 4 fl oz
ChildLife provides a complete line of nutritional supplements designed specifically for infants and children. Our experience with infants and children's healthcare is extensive. First Defense Immune Boost 4 fl oz
Dr. Murray Clarke, who founded the company and developed the product line, has been specializing in pediatrics in his homeopathic and nutritional clinic for the past twenty years. The ChildLife Essentials line is literally the product of this experience. The ten products we offer are those, which have proven to be the most important, and the most effective in supporting healthy development and promoting natural immune strength in infants and children. First Defense Immune Boost 4 fl oz
ChildLife Essentials are made from the highest quality natural ingredients. There are no artificial additives in any of our products. No artificial flavorings, no artificial sweeteners, and no added colorings. The great taste of the products makes taking nutritional supplements an easy part of a child's daily routine. First Defense Immune Boost 4 fl oz
These products are sold in natural stores, health food stores and pharmacies throughout America. Internationally, they are distributed in Taiwan. Singapore, China, Philippines, Indonesia, Thailand, Ireland, Croatia, Poland, Romania and New Zealand. First Defense Immune Boost 4 fl oz
VitaDigest Supplement Facts | ||
Serving Size: One Teaspoon (5 ml) | ||
Servings Per Container: 24 | ||
Amount Per Serving | % Daily Value | |
Calories | 8 cal | |
Total Carbohydrate | 2 g | <1%**? |
Olive Leaf Extract (olea europea) | 80 mg | * |
St. Johns Wort (Hypericum perforatum) (leaf & flower) | 20 mg | * |
Larch arabinogalactan (larix occidentalis) (tree) | 15 mg | * |
Zinc gluconate | 3 mg | 20% |
Colloidal Silver | 12 ppm | * |
* Percent Daily Values are based on a 2000 calorie diet. *Daily Value not established. |
The products and the claims made about specific products on or through this site have not been evaluated by the United States Food and Drug Administration and are not approved to diagnose, treat, cure or prevent disease. The information provided on this site is for informational purposes only and is not intended as a substitute for advice from your physician or other health care professional.
Products image are NOT always exactly as shown on the photographs. We try to be as accurate as possible with photos. Please check the Title carefully before ordering.
All models are 18 yrs or older.
Copyright © 2002-2017 VitaDigest.com. All Rights Reserved.


text:
Check out the official trailer for the final book in the Hunger Games Trilogy!
Disclaimer: Mockingjay.co.uk is an unofficial fan site and is therefore unaffiliated with Suzanne Collins, Scholastic, Lionsgate Entertainment or any other party related to the Hunger Games Trilogy.
Website Design by Gregory Porter
"Only I keep wishing I could think of a way to to show the Capitol they dont own me. That Im more than just a piece in their Games."
- Peeta Mellark


text:
Opened 4 years ago
Last modified 16 months ago
#22377 assigned enhancement
Add filter to wp_update_plugins() — at Initial Version
Reported by: | rmccue | Owned by: | |
Milestone: | Awaiting Review | Priority: | low |
Severity: | minor | Version: | 3.4 |
Component: | Upgrade/Install | Keywords: | has-patch |
Focuses: | Cc: |
Certain plugins (paid plugins, etc) may not wish to use WordPress' built-in
upgrading functionality. As such, they may implement their own update checking
code. WordPress' code runs every 12 hours via wp-cron, so hooking into the
native system for checking would be the best way to avoid duplicating tonnes of
Currently, filtering this result is somewhat unintuitive. There are no filters
in wp_update_plugins() to filter the result of the API call, so you have to
fall back to pre_set_site_transient_update_plugins. WordPress, however, sets
this transient twice,
once before updating] "to prevent multiple blocking requests if request hangs"
and then once afterwards with the actual result.
This is only a minor inconvience, but it appears to be somewhat fragile.
Ensuring that a plugin's custom code doesn't run on the first set_transient()
requires setting a flag, and this is prone to breaking if wp_update_plugins()
changes internally.
Introduce a new filter to change the API result before saving. This filter
should only run once, with the actual result of the API call.
Patch attached.


text:
ISLAND FALLS — Maine State police say four young children were injured and a horse was killed when a car driven by a Sherman man struck their horse-drawn buggy in Island Falls.
The children – ages 9, 8, 7 and 5 – are members of the small Amish community in Aroostook County and were transporting wood.
They were taken to the hospital in Houlton with non-life threatening injuries after the accident at about 2:30 p.m. Monday.
Police say 62-year-old Stephen Porter was driving west on U.S. Route 2 when the buggy emerged from behind a thick tree line into the road without stopping to check for traffic.
Porter could not see the buggy and struck the horse, sending the buggy into the air and ejecting the children.
The crash remains under investigation.
Were you interviewed for this story? If so, please fill out our accuracy form


text:
Mini Meals – smaller portion meals, upto 220g
A pastry base topped with raspberry, plum and apple jam layered in delicious sponge and served with custard.
Celery Free, Mustard Free, Soya Free, Yeast Free, Beef Free, Tomato Free, Garlic Free, Onion Free, Mushroom Free, Alcohol Free, Fish Free, Shellfish Free, Crustacean Free, Mollusc Free, Sulphites Free, Cheese Free
Cooking Guidelines (all ovens may vary): Pre-heated oven 160 C/325 F/Gas 3-4. If fan assisted 140 C/275 F. Typically 20/25 minutes or until piping hot. Lid on.
Cook from frozen. Position the plastic container in the centre of the microwave oven. Cook on FULL power.
If you use our Mikrofix microwaves then this meal requires number 27
Store at -18 C, do not refreeze once thawed.
water, WHEAT flour (with calcium, iron, niacin, thiamin), sugar, vegetable oils (rapeseed, palm), jam (9%) [glucose syrup, liquid sugar, apple pulp, plum puree, acidity regulators (citric acid, sodium citrates), seedless raspberry puree concentrate, colour (anthocyanins), gelling agent (pectins), modified maize starch, whey (MILK) powder, palm fat, dried EGG powder, cornflour, MILK proteins, raising agents (E450(i), E501(ii)), glucose syrup, salt, dehydrated potato, natural flavourings, colour (E160b)
Made in a factory that does not handle nuts. Not guaranteed free of nut traces. Allergy Advice: For allergens see ingredients in CAPITALS
Please note that ingredients may change from time to time, meaning more than one recipe could be in circulation. Whilst every effort has been made to ensure all variants are detailed here, please refer to the product labels for the exact specification of any individual dish. If you have any concerns regarding ingredient changes, please ask when placing or receiving your order.


text:
I thinking about doing this after I seen my buddys truck with one but he had someone put it in for him. I will be putting this in my car myself. I think this would go good with my 2 12's in the back. I need some Ideas I want a racing look on the out side but a nice new look on the inside. Any Ideas would help.
assuming you are talking about an in-dash flip out screen, it should be pretty much like installing a din head unit. i think most classic guys get a spare bezel they can cut to fit and keep the other original. that should be it as far as making it fit, but I dont really know classics very well, is there a part of dash that hangs over the stereo? if you you will probably want to get some abs plasic or thin sheetmetal to make some sort of gap cover so you can have it not flush with the dash so the screen is viewable


text:
Onetime long shot Ted Cruz won the Republican nomination in a U.S. Senate race in Texas on Tuesday, providing tea party activists with renewed momentum in what they said was their biggest victory of the year.
Cruz, a 41-year-old former Texas solicitor general and a first-time candidate for elective office, is the tea party's first bona fide star of the 2012 campaign: a charismatic speaker with an up-by-the-bootstraps biography who upended the Republican establishment in the nation's largest red state.
With most precincts reporting, Cruz held a lead of 55 percent to 45 percent over Lt. Gov. David Dewhurst, a powerful GOP figure who spent freely from his vast personal fortune and had endorsements from most of the state's influential Republicans, including Gov. Rick Perry.
Tea party leaders hailed Cruz's performance as a sign of the movement's political maturation. After bursting onto the scene in 2010, the tea party this year suffered defeats in a few Senate primaries, appeared divided in several GOP contests, and before Tuesday mustered just one clear victory — in Indiana, where state Treasurer Richard Mourdock ousted 36-year Sen. Richard G. Lugar (R-Ind.), whose missteps contributed to his primary defeat.


text:
MESOPELAGIC (and some BATHYPELAGIC)
A. DEEP PELAGIC FISHES -- Oregon/California |
Midwater trawl Lanternfish accidentally caught with Alvin (2006) and Viperfish caught with midwater trawl during Alvin expedition (2007) |
These are fish that we caught either as our deep-sea bottom otter
trawl rose, or by our midwater trawl (figure, left top).
Most are MESOPELAGIC (about 200-1000m), but some of these species
are also found in the deeper BATHYPELAGIC zone (below 1000m) and
ABYSSOPELAGIC (below about 3000m, but poorly defined). The deepest
known fish is at about 8300m (see Bathyal and Abyssal
Fishes page). Other pictures below were obtained from submersibles.
Thanks to Ruben Pohl for the depth ranges, which are listed first
as peak abundance, followed by record depths.
TO FIND almost any FISH SPECIES in the world, go to
FISHBASE. The
Fishbase site (mostly non-English) has many pictures of fishes from
all depths, and can be searched by fish family names (in English). |
Crested Bigscale Poromitra crassiceps (Primary depth 300-3200m) |
Daggertooth Anotopterus pharao/nikparini ( 500-2700m; young rise to 20m ?) |
Barreleye Macropinna microstoma ( mainly 500-1000m) |
Barreleye, top view |
Snipe eel Nemichthys scolopaceus (mainly 50-1500m) |
DragonfishAstronesthes martinii
(depth 350-1500m) from the Red Sea; photos provided by Ruben Pohl of Vienna,
Austria: |
Headlightfish Diaphus theta ( 0-200 at night; 300-1000 during day; max 1687m?) |
Northern Pearleye Benthalbella dentata ( mainly 400-1000m) |
Dragonfish Tactostoma macropus ( 50-200m at night; 300-1600m during day; max 2000m?) |
Fangtooth Anoplogaster cornuta (mainly 300-2000m; larvae 0-200m) |
Anglerfish |
Viperfish Chauliodus macouni ( mainly 250-950m; max 4231m?) |
Another viperfish Note light organ behind eye |
Ventral light organs on viper and lanternfish |
Viper/Dragonfish gape |
.. Stout Blacksmelt and "gel" layer Pseudobathylagus (Bathylagus) milleri (200-1400m; max. 6600m !) --see related fish just below and notes below**-- |
2009 pictures (Monterey Bay) | |||||
Northern Lampfish Stenobrachius leucopsarus ( mainly 31-1189m; max 3400m?) |
Hatchetfish Argyropelecus? |
Hatchetfish's large mouth |
Hatchetfish's ventral (belly) light organs or photophores |
Witch eel Facciolella sp. (935m?) |
Slender Blacksmelt Bathylagus pacificus (0-7700 m! usually 600-800m) --see related fish just above and notes below** |
**Our Research: In the 1980s we
found that some midwater fish have buoyant gelatinous layers (ABOVE
2 rows, RIGHT: layer just under skin in the blacksmelt cross-section).
They also have very low protein content in muscles. These are probably
adaptations to an energy-poor environment; it also means these are not very
good for human consumption. Our publications on midwater fish anatomy
and biochemistry: --Siebenaller, J.F., P.H. Yancey (1984). The protein composition of white skeletal muscle from mesopelagic fishes having different water and protein contents. Mar. Biol. 78: 129-137 --Yancey, P.H., R. Lawrence-Berrey, M. D. Douglas (1989). Adaptations in mesopelagic fishes. I. Buoyant glycosaminoglycan layers in species without diel vertical migrations. Mar. Biol. 103: 453-459 --Yancey, P.H., T. Kulongoski, M.D. Usibelli, R. Lawrence-Berrey, A. Pedersen (1992). Adaptations in mesopelagic fishes. II. Protein contents of various muscles and actomyosin contents and structure of swimming muscle. Comp. Biochem. Physiol. 103B: 691-697 |
B. OTHER DEEP PELAGIC ANIMALS--Oregon, California/NE Pacific |
Gelatinous zooplankton--Salps, larvaceans, siphonophores;
250-880m deep. Most are Larvacea
(taken from submersible camera). I took the leftmost-one with Alvin's video at 880m.
Midwater/mesopelagic salp* caught with net
Unknown--rear (left) tentacles were moving
Jelly or hydrozoan medusa* at 511m
Atolla wheeltop
jelly (mesopelagic) |
VAMPIRE SQUID. See MOLLUSC
PAGE for details
Cirrate octopods. See MOLLUSC
PAGE for details
Chiroteuthis calyx=GLASS SQUID with intact feeding arms! See MOLLUSC
PAGE for details
Teuthowenia-type squid
(seen from Alvin at 880m) |
PETER BATSON'S Photos **These great pictures are not mine, but are copyrighted and courtesy of PETER BATSON , marine researcher and author of DEEP NEW ZEALAND-Blue Water, Black Abyss (2003). Please do NOT use the pictures without his permission! See his website: Exploretheabyss.com. |
Teuthowenia, an odd midwater squid
Ever- |
Hydrolagus, a
chimaera (relative of sharks) |
FOR MORE DETAILS, see these WEB SITES and REFERENCES |
A nice overview of common and interesting deepsea animals (with some of my pictures) can be found at Sea and Sky. The Monterey Bay Aquarium Deepsea page has excellent pictures |
Pictures and descriptions of
the strange bathypelagic Anglerfish
are at Ramkat's website. For pictures and information on the giant oarfish, click here |
A nice site with drawings and animations of deep pelagic animals (vampire squids, anglerfish, etc.) can be found at CYBERSPACE: Below the EDGE. The Monterey Bay Aquarium Deepsea page has excellent pictures of real pelagic animals in their habitats, including Gulper Eels, jellies, ctenophores, etc.. | |
Pictures of the bizarre midwater Vampire squid can be found at Smithsonian Magazine. | |
See the UCSB Bioluminescence or the Harbor Branch Oceanographic Bioluminescence page for more information on biological light production, including in the deep sea. |


text:
These are some etchings my partner Catherine Stack and I have been working on lately. I just finished a comic strip for Atomic Books' FCBD paper Mutant #3. When I get the o.k. I will post that soon! Sorry for the lack of updates, working full time, lots of band stuff and not enough time for sleep has made working on my art difficult!!
Saturday, April 2, 2011
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Answer by sal jefferson · Sep 03, 2014 at 09:31 AM
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Answer by sal jefferson · Sep 03, 2014 at 10:06 AM
Answer by sal jefferson · Sep 03, 2014 at 10:31 AM
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Answer by Adam Tirapelle · Sep 03, 2014 at 08:24 AM
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