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ABC IMMUNOHISTOCHEMISTRY PROTOCOL
1) Cells or frozen -sections: fix in acetone (RT) 5 min or 2% paraformaldehyde (RT) 5 min (empirically determined for each antigen), then rinse in PBS X 3. Paraffin sections: deparaffinize.
2) Blot off excess PBS; etch around section with diamond pen; label slide with pencil; place in humidity chamber for all steps.
3) If needed, block endogenous peroxidases (all tissues). Dip slides in water, then incubate in MeOH/H202 for 30 min at RT. PBS rinse X 3.
4) Incubate with blocking serum (same species as secondary antibody) for 20 min.
5) Blot excess.
6) Incubate with primary antibody overnight.
7) PBS "sprits" (use a pipette to gently rinse off antibody from slide), then 3X 2 min each (approx. 150 ml each). Blot excess.
8) Incubate with secondary antibody 1 hour. Prepare ABC reagent (needs to polymerize at RT for at least 30 min prior to use).
9) PBS sprits, then 3X 2 min each. Blot excess.
10) Incubate with ABC reagent for 40 min.
11) PBS sprits, then 3X 2 min each.
12) Incubate with freshly prepared DAB/H202 solution 4 (monoclonal) to 6 (polyclonal) min. These incubation times are guidelines, and can be determined experimentally to optimize signal to noise.
13) PBS sprits, then 3X 2 min each. Handle and dispose of waste DAB appropriately (it's carcinogenic).
14) Stain with hematoxylin and eosin and mount.
NB:
1) Never allow section to become dry once you begin experiment. After manipulating slide, keep in humidity chamber.
2) Plastic containers (Tissue-Tek Staining Set, S7626-12), slide holders and trays (S7626-3), etc. useful for washes/staining purchased from American Scientific Products.
3) Humidity chamber is a plastic box with elevated strips suitable for holding slides. Bottom is filled with water.
4) For problems with nonspecific staining, etc, see suggestions in VectaStain Kit. For some antibodies, for example, you may need to increase the % of SERUM/BSA in the blocking solution and/or the diluent of the primary antibody.
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