Revised 04/2007 bmcl
Immunohistochemistry of Tissue Sections
Date: ________________
Experiment: ____________________________
Note: This protocol describes AP and HRP staining, not fluorescence. Use shaker for all washes and incubations for
free-floating sections.
1. Pull appropriate tissue sections and put into staining dishes.
2. Wash at least 3X for 5 min in PBS.
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3. Wash 1X for 5 min in 1.6 ml 30% H2O2 in 100 ml PBS (0.5%) to inactivate endogenous peroxidases.
4. Wash at least 4X for 5 min in PBS.
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5. Wash at least 3X for 10 min in 0.1M Tris-Glycine (0.1M glycine, pH to 7.4 with Tris Base).
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6. Wash at least 4X for 5 min in PBS.
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7. Wash at least 4X for 10 min in 4% Blotto, 0.2% Triton-X 100 in PBS (omit Triton for membrane-bound antigens).
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8. Prepare primary antibody: ____________________________________.
Dilute 1:____ in 4% Blotto, 0.2% Triton-X in PBS.
Need _____ ml so use _____ l anti____________ in ______ ml Blotto.
9. Incubate 72 hrs at 4C (can also incubate overnight at RT but the background will be higher).
10. Wash at least 5X for 5 min in 4% Blotto, 0.2% Triton-X.
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11. Prepare secondary antibody: __________________________________.
Dilute 1:1000 in 4% Blotto, 0.2% Triton-X in PBS.
Need _____ ml so use _____ l anti____________ in ______ ml Blotto.
12. Incubate in secondary for 60 min at RT.
13. Near end of secondary incubation, prepare standard ABC reagent (if biotinylated secondary):
Add 2 drops A per 10 ml PBS and mix. Add 2 drops B and mix. Let sit 30 min before use.
14. Wash at least 5X for 5 min in PBS.
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15. Apply ABC reagent for 60 min at RT (if biotinylated secondary).
16. Wash at least 5X for 5 min in PBS (if biotinylated secondary).
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17. Apply DAB- H2O2l 30% H2O2, 1ml 20X DAB, 19 ml PBS) for ~5 min at RT (determine optimal time
experimentally).
18. Wash at least 3X for 5 min in PBS.
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19. Mount sections onto subbed slides, dehydrate, and coverslip with DPX.