Mouse Anti-_BrdU in paraffin embedded sections
- Prewarm 2N HCl at 37 C. and prewarm 0.1M Tris PH 7.5.
- Deparaffinize 20minX2. Rehydrate slides to ddH2O.
- Incubate slides in 2N HCl at 37 C for 15 min.
- Rinse in ddH2O 5min X 4.
- 0.05% Trypsin in 0.1M Tris PH 7.5 at 37 C for 15 min.
2.5% trypsin 1ml + 29ml in 0.1M Tris PH 7.5
- .Wash in PBS 5min X 2.
- Block endogenous peroxidase with 3% hydrogen peroxide in PBS) for 15 min.
- Wash in PBS 5min X 3.
- Block non-specific proteins with 10% normal goat serum in 0.5% PBST (PBS+0.5% Tween 20) for 30-60 min at RT.
- Incubate with mouse anti BrdU 1:200---1:400 in Blocking buffer (5% goat serum in 0.1% PBT ) O/N at 4 C.
- Wash slides with PBST 5min X3.
- Incubate with 1:200 Biotinylated goat anti-mouse IgG (Vector) 1 hour at RT.
- Make up ABC ( 30min before use) ABC 2.5ml (1:50)PBS+ 1drop of A + 1drop of B( 1drop=50ul )
- Wash slides in PBST for 5minX3.
- Incubate with ABC for 45 min.
- Wash in PBSTfor 5min X3.
- Wash in 0.1M Tris pH 7.5.( 0.1M Tris 7.5 & 0.3 M NaCl )
- Color developing:
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 in air. Mount the slides with mounting medium.