Edited by Chang Zhu
1. Section paraffin blocks at 4 microns and float on a water
(deionized or distilled) bath at 45 °C.
2. Pick up flattened sections, and place on slide and
dry upright overnight at room temperature. Store at room temperature.
3. Deparaffinize in xylol, 3 x 3 minutes.
4. Rehydrate in decreasing grades of ethanol: 100%, 96%, 70%, 50%, 3 minutes each.
5. Block endogenous peroxidase activity by incubating slides for 10 minutes in freshly made 0.3% H202 in methanol.
6. Rinse in water then with PBS.
7. (Optional)
- Incubate sections with 0.1% Trypsin in 0.1% CaCl2 pH 7.6 for 10 minutes at room temperature.
- Wash with PBS, 3 x 3 minutes.
8. Cover the sections with properly diluted primary antibody. Incubate over-night in a humidity chamber at room temperature or 4 °C.
9. Rinse 3X with PBS.
10. Cover the sections with properly diluted secondary antibody (biotinylated). Incubate 0.5-2 hours at room temperature or 4 °C.
11. Wash with PBS, 3 x 3 minutes.
12. Apply Strepavidin/HRP and incubate at room temperature for 30 minutes.
13. Wash with PBS, 3 x 3 minutes.
14. Stain with DAB.
15. Wash with running tap water for 3 minutes.
16. Counterstain with Mayer's hematoxylin, 2 minutes.
17. Wash with running tap water for 2 minutes.
18. Dehydrate through 4 changes of increasing grades of alcohol to 100%.
19. Clear in 3-4 changes of xylene.
20. Mount.
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