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