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OPTIMAL POSTFIXATION WASHING TIME FOR ULTRASTRUCTURAL DEMONSTRATION OF GLUCOSE 6-PHOSPHATASE ACTIVITY

Although tissues being prepared for enzyme cytochemistry are routinely washed with buffered solution to remove glutaraldehyde or formaldehyde, the possibility that postfixation washing causes change in the activities of the enzymes being studied has received only limited attention (2, 6). In the ultrastructural demonstration of glucose 6-phosphatase (G-6-Pase) activity, periods of postfixation washing varying from 1 to 24 hr have been used (4, 7-12, 15-18). In the course of investigating G-6-Pase cytochemistry, I have observed a considerable decrease in activity as a result of a long postfixation washing. This prompted an investigation to determine the optimal washing time of fixed tissues for the cytochemical demonstration of G-6-Pase activity. Male DDD mice, 2-3 months of age, were used. They had free access to food and water prior to the experiments, and were anesthetized by intraperitoneal injection of Nembutal. Transparenchymal perfusion of the liver, modified from those reported previously (10, 19), was carried out using a 0.3-mm hypodermic needle and 5-ml syringe containing 2% redistilled glutaraldehyde (5, 13) buffered with 0.1 M cacodylate at pH 7.0 at room temperature (20-22#{176}C). The abdomen was opened to expose the liver and intestine. The perfusion needle was inserted into the parenchyma of the liver at the edge of the left lobe. A branch of the superior mesentenic artery and vein was cut open with scissors and the perfusion was started immediately at a flow rate of 0.05 mI/sec. The perfusion was continued for exactly 1 ‘22 mm, after which the liver became hard and pale. The tissue within 1 cm of the perfusion needle was discarded to eliminate destroyed tissue. The animals were divided into two groups. Small (0.1-0.2 g) blocks of the fixed liver from one group were immediately frozen on Dry Ice to stop the effect of glutaraldehyde on enzyme activity. The blocks were weighed, cut at 30 z using a freezing microtome, immersed and washed immediately in 0.3 M sucrose containing 0.01 M cacodylate (pH 7.0) at 4#{176}C for 5, 15 or 30 mm and 1, 2, 4, 16 or 24 hr, respectively. The fixed tissues from the other group were quickly cut at about 1-mm thick slices in cold sucrose solution, washed in the same manner as the tissues of the first group and then cut at 30 . Sections prepared thus were immediately used for biochemical, cytochemical and photometric analysis. The volume of the washing solution was 100 ml/0.i g of tissue and the solution was changed three times, after 30 mm, 1 hr and 2 hr. Both 3O-M sections and i-mm slices were immersed and continuously shaken for the first 4 hr of the washing. Biochemical methods: Washed sections were collected by brief centrifugation at 4#{176}C and the resulting 0.1-0.2 g pellets were homogenized at 0-4#{176}C in 10 volumes 0.25 M sucrose containing 0.01 M cacodylate (pH 7.0) in a Potter-Elvehjem-type homogenizer for 5 mm at 3000 rpm. The homogenates were immediately used for assay of G-6-Pase according to the method described by Leskes, Siekevitz and Palade (12). Incubation time was 20 mm. The inorganic phosphate released was determined by the method of Allen (1). Proteins were estimated according to the method of Lowry et al. (14). The activity was expressed as micromoles of phosphorus liberated per milligram of protein per 20 mm, and the values were expressed as a percentage of the highest ones (those obtained with the 5-mm washing). Cytochemical and photometric methods: The washed sections were incubated in Wachstein and Meisel’s medium for the demonstration of G-6-Pase (20) for 15 mm at room temperature (20-22#{176}C). The sections were washed in 0:3 M sucrose solution for