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Star Republic: Guide for Biologists

Staining --- formalin-fixed section

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.