3.3.1. Heat Treatment
1. Place the slides with adhered tissue on a heat-block at 105°C for 5 to 120 s to stabilize the cells or tissues (see Note 6).
1. Place the slides in a solution of 4% paraformaldehyde in PBS for 4 h at room temperature. Using the recommended Coplin jars or staining dishes facilitates these steps.
2. Wash the slides once with 3X PBS for 10 min, agitating periodically with an up and down motion.
3. Wash the slides with 1X PBS for 10 min, agitating periodically with an up and down motion. Repeat twice with fresh 1X PBS.
4. At this point, slides with adhered tissue can be stored at -70°C until use. Before storage, dehydrate with 100% ethanol.
5. If biotinylated probes or peroxidase-based color developments are to be used, the samples should further be treated with a 0.3% solution of hydrogen peroxide in PBS.
6. Incubate the slides overnight—either at 37°C or at room temperature, then, wash the slides once with PBS.
7. If other detection systems are to be used, proceed directly to the proteinase K digestion.
3.3.3. Proteinase K Treatment
Very Important! Two alternative methods for proteinase K digestion are described.
188.8.131.52. Proteinase K Digestion Method A
1. Treat samples with 6 |g/mL proteinase K in PBS for 5 to 60 min at room temperature or at 55°C (no doubt this represents quite a range).
2. After 5 min, look at the cells under the microscope at x400.
If the majority of the cells-of-interest exhibit uniform-appearing, small, round "bubbles" or "blebs" or "peppery dots" on the cytoplasmic membrane, then stop the treatment immediately with step 3. Otherwise, continue treatment for another 5 min and re-examine.
3. After proper digestion, heat slides on a block at 95°C for 2 min to inactive the proteinase K.
4. Rinse slides in 1X PBS for 10 s.
5. Rinse slides in distilled water for 10 s.
184.108.40.206. Proteinase K Digestion Method B
1. Prepare four or five extra slides of the specific tissue in question. It is especially helpful if the slides are successive sections or very similarly prepared, for the morphology of the various slides must be closely compared later.
2. Prepare an equal number of serial dilutions of proteinase K solution, for example, over the range of 1-6 mg/mL.
3. Decide upon a standard time and temperature for digestion, for example, 15 min at 37°C.
4. Treat the slides in the serial solutions for this standard period.
5. Stop the digestion by heating slides on a block for 2 min at 95°C.
6. Counterstain the slides with hematoxylin or other appropriate stain.
7. Observe slides under a high-power light microscope and look for any morphological change in the cells.
8. Choose the highest concentration of proteinase K that did not result in significant change as the optimized concentration.
9. If all the slides showed change, repeat process with lower concentrations of pro-teinase K or shorter incubations.
10. If none of the slides showed change, increase concentrations or incubation times and repeat procedure until the proper conditions are found.
11. Once the optimized digestion is determined, use these conditions to process all slides of that particular tissue, fixation method, and thickness of section. Any change in the latter three parameters necessitates re-optimization of the digestion procedure (see Note 7).
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