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ABC-HRP in Paraffin Embedded Sections

1. Deparaffinize 20minX2. Rehydrate slides to ddH₂O.
   (100% ETOH 2min. 95% ETOH 2min. ddH₂O 2min X2).
2. Wash in PBS 3min X2.
3. Block endogenous peroxidase with 3% hydrogen peroxide in PBS for 15 min.
4. Wash in 0.1% PBST (0.1% Tween 20 in PBS) 2min X3.
5. Block non-specific proteins with 10% normal serum in 0.5% PBST (0.5% Tween 20 in PBS) for 30 60min at RT.
6. Incubate with 1st antibody in 5% normal goat serum in 0.1% PBST O/N at 4°C.
7. Wash slides with PBST 5min X6.
8. Incubate with 1:200 Biotinylated 2nd antibody (Vector) 1 hour at RT.
9. Make up ABC (Vector):
   ABC 2.5ml PBS+ 1drop of A + 1drop of B( 1drop=50ul )
10. Wash slides in PBST for 5minX6.
11. Incubate with ABC for 45-60 min.
12. Wash in PBST for 5min X4.
13. Wash in 0.1MTris pH 7.5 and 0.3M NaCl 5minX2.
14. Color developing:
    A. DAB: One set tablets (Sigma) to 5ml ddH₂O.
15. Add DAB to slides for 2-10min.
16. Wash with ddH₂O several times.
17. Counter-stain with Hematoxylin for 1 min.
18. Wash and dehydrate. Mount the slides with mounting medium.

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