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Protocols
cryosectioning,
avidin-biotin, sample fixation |
Buffers
tissue fixation, permeabilization,
PBS ... |
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Links
to
microscopy and histochemistry resources
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| avidin
- biotin & dab immunohistochemistry |
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1. Deparaffinize
sections
2. Rehydrate In steps through to distilled water
(dH2O).
3. Immerse sections in 0.5% v/v hydrogen peroxide/methanol
for 10 minutes.
4. The antigen
retrieval protocol can be carried out at this
time, if this procedure is deemed necessary.
5. Wash the tissue sections in two changes of dH2O
(2 x 5 minutes).
6. Immerse the sample slides in 50mM Phosphate
Buffered Saline (PBS) 2 x 5 minutes.
7. Cover sections with blocking solution.
8. Remove the blocking solution.
9. Quickly circle the tissue section with a PAP
pen (the tissue section should not be allowed to
dry).
10. Add primary antiserum diluted in blocking solution
and incubate overnight at 4ºC (or 60 minutes
at 25ºC).
11. Wash in PBS buffer for 3 x 5 minutes.
12. Remove excess PBS buffer and incubate sections
with biotinylated secondary antibody diluted in
blocking reagent; incubate for 30 minutes at 25ºC.
13. Wash in PBS buffer for 3 x 5 minutes.
14. Remove excess PBS buffer and incubate sections
with ABComplex/HRP
for 30 minutes at 25ºC.
15. Wash in PBS buffer for 3 x 5 minutes.
16. Develop with 3 3’ diaminobenzidine
tetrahydrochloride (DAB)
17. Rinse slides in gently running tap water.
18. Counterstain with Haematoxylin.
19. Dehydrate, clear and mount sections.
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