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SUMIC: A Simple Ultrafast Multicolor Immunolabelling and Clearing Approach for Whole-Organ and Large Tissue 3D Imaging

High-resolution whole-organ imaging of cleared tissues captures cellular and molecular insights within the intact tissue and tumour microenvironments. However, current immunolabelling and clearing methods are complicated and time-consuming; extending to several weeks. Here, we developed Simple Ultrafast Multicolor Immunolabelling and Clearing or SUMIC, a method that enables multicolor immunolabelling and clearing of whole murine organs and human tissues within 2 to 2.5 days. Moreover, SUMIC is simple, robust, non-hazardous and versatile comprising antigen retrieval, permeabilization, collagenase-based digestion, immunolabelling, dehydration, and clearing. SUMIC permits quantitative and singlecell resolution analysis and detection of rare cells in whole organs, for example, round αSMA positive cells in the thymus. Upon volumetric imaging, SUMIC-processed samples retain normal tissue architecture and can be used for paraffin-embedding and histology. We employed the SUMIC method for whole-organ mapping of lymphatic vessels across different ages and organs. This analysis revealed the expansion of lymphatic vessels in endocrine tissues but not in any other organs with aging. Hence, SUMIC will accelerate discoveries compared to other whole organ imaging pipelines.


Introduction
Advanced 3D imaging approaches provide critical insights into the fundamentals of highly complex biological processes during organ development, regeneration, repair and pathology (1-7). To advance our understanding of such processes, it is vital to generate accurate cellular, and molecular cartography of healthy, aged, diseased and malignant tissues is vital (8)(9)(10)(11). The 3D analysis of intact biological specimens reveals essential structural and spatial information necessary for the understanding of the crosstalk among various cell types in the tissue context (9,(12)(13)(14)(15)(16). Moreover, deciphering the organization of the nervous and vascular systems relies on a whole organ approach (12,(17)(18)(19)(20). This treasure trove of information comes at the expense of detailed and complicated requirements for time-consuming sample processing, imaging, data storage, analysis and interpretation, which can be met by a range of recent technical developments.
The recent development of tissue clearing, light sheet microscopy and large dataset analysis has made it possible to image large volumes at single-cell resolution (13,(21)(22)(23). However, current protocols for immunostaining and whole organ clearing are often time-consuming, elaborate, expensive, and organ-specific (24)(25)(26)(27)(28)(29)(30)(31)(32). The timeconsuming nature of the whole organ and large tissue imaging is the main obstacle to overcome before efficiently applying this crucial technique to understand biological complexity and interrogating cellular interactions within tissues and organs. Other disadvantages include the requirement for transgenic mice that express fluorescent reporters and the use of toxic or corrosive chemicals that might be detrimental either to the microscope or researcher's well-being (11,(33)(34)(35).
Therefore, it is essential to develop a rapid method for whole organ and large tissue multicolor immunostaining coupled with a technique applicable for diverse tissues while preserving endogenous fluorescence. Here we devised a new robust and straightforward method-Simple Ultrafast Multicolor Immunolabelling and Clearing or SUMIC which permits immunostaining and clearing of multiple whole organs and large tissues in a record time of 2.5 days, and also overcomes other limitations of current methods.

SUMIC: an ultrafast immunostaining and clearing method for murine organs and human tissues
In SUMIC we set out to develop a platform that is able to attain a multicolor immunolabelling together with excellent cellular morphology, tissue architecture and transparency across multiple murine organs and human tissues. Each of the 10 steps were optimized to enable completion in the shortest possible time (Fig. 1). First, we combined paraformaldehyde and glutaraldehyde for rapid fixation (Step 1). Samples were bleached with hydrogen peroxide in methanol to eliminate heme and other persistent chromophores (Step 2). Urea facilitated antigen retrieval, and Triton X-100 increased the penetration depth of antibodies into tissues while maintaining tissue architecture (Step 3). Collagenase-digestion of the extracellular matrix significantly improved the penetration rate of the antibodies while keeping the structure of the tissues intact (Step 4). To obtain good staining, only antibodies labelled with small, bright fluorochromes with high stability and good tissue penetrance, such as Alexa Fluor dyes, were used (Step 5). Triton X 100, a mild detergent was included for blocking, staining and washing the organs during this step. Incubations with antibodies and their washings were performed at 37°C to reduce the time requirement further.
Dehydration is a crucial step that precedes tissue clearing and RI matching (Step 6).
Dehydration using isopropanol preserved all fluorochromes and resulted in less tissue shrinkage compared to ethanol. Ethyl cinnamate (ethyl 3-phenyl-2-propenoate; ECi) is non-toxic and has been utilized for tissue clearing (11). Poly(ethylene glycol) methyl ether methacrylate (PEGM) has also been used as a component of tissue clearing solutions (26). For SUMIC ECi (80%) and PEGM (20%) were combined. This combination of ECi and PEGM cleared all the tested organs and tissues within 2 hours (Step 7). ECi was used for RI matching for imaging (Step 8). Starting from collecting the organs to image acquisition, SUMIC protocol for multicolor antibody labelling and tissue clearing required up to 2.5 days with conjugated antibodies (Fig. 1). SUMIC also includes a rendering and analysis pipeline (Step 9).
We have successfully validated SUMIC across multiple murine organs, including lung, heart, kidney, liver, gut, spleen, thymus, prostate gland, seminal vesicle and endocrine glands (Fig. 2, Fig. 3, Fig. S1 and Movie 1-6). Notably, a few organs, such as liver, demonstrated a slightly brownish yellow colour even after the completion of the clearing process (Fig. 2D); however, this had minimal impact on the penetration depth of the light sheet. We selected ~40 of antibodies to test and validate SUMIC method across multiple organs and tissues. We selected antibodies for the cell and matrix molecules expressed across different organs. The cell markers defining vascular and tissue microenvironments, including endothelial cells, pericytes, mesenchymal cells and immune cells, were used. Endothelial cell markers; Endomucin (Emcn), Endoglin (CD105), CD31 also known as PECAM1 and CD102 (ICAM2); a-SMA staining was used for artery quantifications, and pericytes were identified by PDGFRb and NG2.
Lymphatic vessels were analyzed by LYVE1 and Prox1. We provide full information for each of these antibodies and their working conditions (Table S1). With these cell surface markers, SUMIC enabled 3D imaging of blood vessels and associated niches across several organs and endocrine glands (Fig. 2, Fig. 3, Fig. S1 and Movie 1-6).
Thus, the SUMIC method provided the flexibility of usage across multiple organs and tissues. Troubleshooting tips relevant to the SUMIC method can be found in Table S2, and the methods section of the paper. In addition to the murine organs, SUMIC permitted ultrafast immunostaining and clearing of human tissues (Fig. 4, A and B).

SUMIC preserves tissue architecture and endogenous GFP/tomato fluorescence
To determine the effect of SUMIC processing on tissue morphology, we embedded SUMIC-processed organs in paraffin and examined the samples previously imaged in 3D by conventional histology. 2D histology showed that SUMIC processed tissues remained accessible to a conventional histological analysis by haematoxylin and eosin ( Fig. S2). Tissue architecture remained unaffected, and different structures, such as blood vessels, were detected in SUMIC processed tissues similar to the control tissues processed by conventional paraffin embedding (Fig. S2). Thus, SUMIC enabled rapid, deep tissue immunolabeling and clearing of intact organs while maintaining tissue architecture.
Endogenous fluorescence loss is a crucial concern for solvent-based tissue clearing medium and other steps during tissue processing. To assess the impact of SUMIC clearing on endogenous fluorescence either Flk1 GFP or CDh5 Cre ERT2 X tdtomato mice was used. Our analysis revealed that the SUMIC processed samples preserved tomato and GFP fluorescence (Fig. 4, C and D).

SUMIC permits single-cell resolution analysis in whole-organs
Light sheet microscopy was used to perform volumetric imaging and thereby produce the whole organ and tissue overviews of SUMIC-processed murine whole organs and human tissues. We next aimed at transitioning from a general overview of the complete tissue to the analysis of specific regions within the tissue at the single-cell resolution. Subsequently, computational surface rendering was applied to the regions of interest, which provides single-cell level information from three-dimensional tile scan images (Fig. 4, E and F). This analysis led to the detection of a small subset of round α-smooth muscle actin positive cells within the cortex region of the thymus in addition to the usual periarteriolar location of these cells (Fig. 4E). Such round α-SMA positive monocyte-macrophage subset has been reported in the bone marrow where these cells preserve primitive hematopoiesis (36). However, such a small subset of α-SMA positive monocytes-macrophages has not been reported to be present in the thymus.

SUMIC enables quantitative imaging of whole organ overviews
Next, we carried out a quantitative analysis of the lymphatic vessels in 3D by LYVE-1 and Prox1 immunostainings on whole organs and endocrine glands. The circulatory system encompasses both the blood vascular and lymphatic vascular systems. The lymphatic system regulates fluid homeostasis, waste clearance and immune responses (37,38). Recent studies have not only revealed age-dependent changes in blood vessels, organs and endocrine glands but also documented the correlation of these changes with the organ function (39). Here, we examined and compared the lymphatic vessels across different endocrine glands. Quantitative analysis of LYVE-1 and Prox1 immunostainings across endocrine glands from young and aged mice demonstrated expansion of lymphatic vessels in ovaries, testis and thyroid glands ( Fig. 5, A and B). In contrast, the lymphatic vessel density remained unchanged across murine organs with age ( Fig. S3).
To understand the relevance of age-related changes observed in these three murine endocrine glands, we analyzed human endocrine tissues. Healthy human testis, ovary and thyroid gland of young (<20 years) and aged (>70 years) individuals (Table S3) were tested for the Prox1 gene by quantitative PCR (qPCR) (Fig. 5C). Likewise, murine endocrine glands, aged human ovary, testis and thyroid glands exhibited an increased Prox1 expression (Fig. 5C). Importantly, analysis of even 4 random human samples of each of these glands showed these significant changes. These results provide evidence for the applicability of SUMIC method, which is not only rapid and robust but also holds tremendous potential in providing new insights and uncovering biological complexities.

Discussion
3D organization of functional networks, such as blood or lymphatic vessels within organs and spatial information of cells within tissues, is important to understand the biological complexity (10,(40)(41)(42)(43). 3D imaging of cleared large tissues or whole organs provides this information (12,14,41,(43)(44)(45). Further, it is a valuable alternative to traditional histology or immunofluorescence of thin tissue sections, both of which lack the crucial 3D information (16,(45)(46)(47)(48). To enable rapid, simplified and high-resolution 3D imaging of whole organs and large tissue samples, we introduce and describe the SUMIC method. SUMIC enables the 3D molecular analysis of intact organs and tissue biopsies at a single-cell and subcellular resolution. A sequence of tissue alterations renders the samples susceptible to rapid and optimum immunostaining and histological staining while preserving cell and tissue integrity. Digestion of extracellular matrix by Collagenase and use of methanol increases antibody penetration.
Collagenase based extracellular matrix digestion and incubation of antibodies at a higher temperature facilitate speed through rapid penetration of the antibodies.
Further, these steps avoid the time-consuming delipidation steps that involve the use of highly toxic substances (44,49). Further, the bleaching step reduces light scattering (49,50). Hydrogen peroxide used for bleaching reduces tissue autofluorescence and is useful, particularly in tissues with high heme content, such as spleen and heart (51).
Bleaching before the immunostaining aids in the retention of these fluorochromes which otherwise are susceptible to bleaching. Thus, SUMIC neither requires an injection of antibodies, nor it relies on transgenic mice expressing fluorescent reporters; it instead uses multicolor immunolabelling by fluorochrome-coupled antibodies.
ECi and PEGM have been used independently in various clearing methods as tissue clearing recipe components or for RI matching due to their inexpensive and flexible nature (11,(51)(52)(53). Here, we combined these two; ECi and PEGM, which facilitates the rapid clearing of larger tissues and organs. After clearing, images can be acquired on different imaging systems such as a laser scanning confocal microscope for singlecell and subcellular resolution or light sheet microscope for whole organ overviews.
Therefore, SUMIC enables multicolor antibody-based immunolabelling, provides good tissue transparency, and preserves cellular morphology and flexibility in microscope usage.
As a result of facilitating high speed, high resolution and flexibility in organ, tissue, fluorochrome and microscope usage, SUMIC has the tremendous potential to make tissue clearing and 3D imaging more widely accessible to a broader audience. SUMIC is a versatile technique that can be used for answering a wide variety of questions throughout the biological sciences. Thus, SUMIC adds to the tools and information needed to investigate complex biological processes that demand spatial and architectural knowledge of cells within tissues and organs.

Mice
Organs and endocrine glands were dissected from adult 8-15-weeks-old male and female wild-type C57BL/6J mice purchased from Jackson Laboratory and bred in-

Tamoxifen treatment
For oral gavage of tamoxifen, tamoxifen (Sigma, T5648) was firstly dissolved in 100% ethanol and then suspended in corn oil to a final concentration of 5 mg/ml. For Cdh5(PAC)-CreERT2; Rosa26-td-Tomato transgenic mice, to induce Cre activity, mice were orally treated with tamoxifen at a dose of 50 mg/kg body weight for three consecutive days. Mice were sacrificed and analyzed two weeks after the last dose of tamoxifen.

Tissue collection and preparation
Freshly dissected mouse tissues were rinsed in PBS (VWR, cat. No. 437117K) and then immediately transferred into ice-cold fixative solution of 4% paraformaldehyde (Sigma-Aldrich, P6148) and 0.05% glutaraldehyde (Sigma-Aldrich 340855) solution for 3 hours. The fixative should be freshly prepared and kept ice cold during its use.
During the dissection of organs, the adjacent muscles and fat should be removed completely as their presence may subvert the successful generation of whole organ overviews during imaging. The samples were then washed with PBS for 3 times at room temperature on a shaker. Each wash lasted for 5 minutes. From our experience, the fixed samples can be stored for up to 4 days at 4° C before moving forward with the bleaching or antigen retrieval and permeabilization. While placing tissues in fixative works well for several murine organs, the perfusion fixation with 4% PFA is recommended to facilitate instant fixation to preserve the life-like state of the tissues. The fixative is hazardous; highly flammable and toxic and must be handled with appropriate PPE and other safety measures.

Bleaching
In larger heme rich organs bleaching was used to achieve maximum downstream organ clarity. Organs were immersed in a methanol gradient of 50%, 80% and 100% for 40 minutes each for dehydration. 100% methanol was changed twice, 20 minutes apart. These dehydration buffers should be ice-cold. Samples were then immersed in 5% (v/v) H2O2 in methanol for up to 3 hours (Sigma-Aldrich Cat No: H1009) for bleaching. Samples were then rehydrated with the reverse methanol concentrations.
Following that, samples were washed three times with PBS for 20 minutes each.
Dehydration for bleaching could also be achieved with Isopropanol that has a less toxic effect. However, methanol is better for removing pigmentation and more effective for delipidation. It also helps with membranes permeabilization and therefore antibody penetration.

Antigen Retrieval, Permeabilization and Collagenase Digestion
The fixed samples were subjected to antigen retrieval, and permeabilization with the

Immunolabelling
For immunostaining, the organs were transferred to 5ml tubes and blocked in 10%

Dehydration and tissue clearing
The immunostained samples were dehydrated in a gradient of isopropanol. Briefly, samples were immersed in a gradient of 30%, 50% and 80% (v/v) isopropanol for 30 minutes each under gentle rotation at room temperature. The organs were then immersed in 100% isopropanol for 1 hour with the 2 changes of isopropanol during this duration. The isopropanol was then completely removed, and the samples were Samples from donors at ages 18-20 years and 70-80 years were selected to compare the young and aged groups, respectively. Histological study was performed to confirm that sample tissues were healthy and lack disease components. Further details on the healthy human tissue samples are provided (Table S3).

Imaging set-up, Image acquisition and Analysis
The cleared samples were imaged on the Miltenyi-LaVision Biotech UltraMicroscope II with the LaVision BioTech ImSpector software (MACs, Miltec bio). The microscope was equipped with a 2X objective lens for zoom body with a manual zoom of 0.63X- Tiles were chosen based on organ size, and images were taken with a 10% overlap in order to stitch them together using Zen Black (version 3.1, Zeiss) software. Organs were imaged through the z-plane and reconstructed in Imaris in order to visualize the maximum intensity projection. In order to visualize the boundary of the organ, the autofluorescence from the 405 channel was converted into greyscale, and the 30% opaque image was manually overlayed with the corresponding TIFF file generated from Imaris. Imaris, Adobe Photoshop and Adobe illustrator software were used to generate, analyze and compile images.

3D surface rendering
To visualize components of the organ, 3D surface rendering was applied using the surface rendering tool in Imaris. First, the whole organ or region of interest (ROI) was defined and selected. Then, the smoothness of the resulting area was set up using the Smooth option. The threshold for background subtraction was manually annotated based on the individual image previewed.

Quantifications of imaging data
To quantify the vascular density of whole organs, quantifications were performed using the Imaris Surface Analysis XTensions tool. To obtain total tissue volume, the 3D crop tool was used and analyzed with the Volume statistics function of Imaris. A single channel lymphatic marker was reconstructed using the 3D rendering surface function, and the tissue volume of the vessels was measured by employing the Surface statistics function in Imaris. The density of the lymphatic vasculature was calculated, dividing the tissue volume of vessels in the numerator by the total tissue volume in the denominator.
Quantification of PROX1 + cell number was done with Imaris software (version 9.2.1) based on single-cell resolution 3D images. Nuclei detection and membrane detection function in the Cell module of Imaris were used to automatically segment and analyze the numbers of PROX1 + cells. Under the Cell Creation wizard of Imaris, PROX1 channel was selected as a source channel for membrane-based detection, and DAPI channel was selected for nuclei-based detection. Nuclei locations were used as seed points for an algorithm performing a cell membrane calculation, which was used to distinguish between the inner and outer boundaries of the cell. The number of PROX1 + cells per tissue volume (mm -3 ) was calculated by dividing the number of segmented cells in the numerator by the total tissue volume in the denominator.

Histology
Mice were sacrificed, and organs (liver, kidney, spleen and heart) were collected. One group of organs were fixed with 10% formalin in PBS. Another group of organs were fixed with 10% formation followed by the steps of SUMIC protocol (dehydration with gradient isopropanol and clearing with ECi). All samples were embedded in a paraffin block and sliced into 5 µm thick sections. Following H&E staining, the samples were imaged by bright-field microscopy (Olympus Co., Japan).

Quantitative PCR
For the analysis of Prox1 expression levels from FPPE blocks of human endocrine tissues, 25 µm sections were generated from FPPE blocks. After deparaffinization,

Statistical analysis
All statistical analyses were performed using GraphPad Prism software (version 9.1).
All data are presented as mean± s.d. The significance of the difference in mean values was determined using two-tailed Student's t-test. The statistical significance was declared when P <0.05. ns: not significant; *: P <0.05; **: P <0.01; ****: P <0.001; ****: P <0.0001. No randomization or blinding was used, and no animals were excluded from analyses. Several independent experiments were performed to guarantee reproducibility of findings.        Tables   Table S1. List of primary antibodies thoroughly tested with SUMIC Table S2. Troubleshooting table   Table S3. Details of healthy young and aged human endocrine gland tissue samples   Step 4 (0.5 h)