Immunostaining in paraffin embedded sections
ABC-AP (Alkaline Phosphatase)
- Deparaffinize 20minX2. Rehydrate slides to ddH2O.
(100% ETOH 2min. 95% ETOH 2min. ddH2O 2min X2).
- Wash in 0.1% PBT 3min X2.
- Block non-specific proteins with 10% goat serum in 0.5% PBT (PBS+0.5% Tween 20) for 30-60 min at RT.
- Incubate with mouse or rabbit antibody in 5% goat serum O/N at 4°C.
- Wash slides with 0.1% PBT 5min X3.
- Incubate with 1:200 Biotinylated goat anti-mouse or rabbit IgG (Vector)in 0.1% PBT for 1hour at RT.
- Make up ABC-AP (Vector) 30min before incubation.
ABC-AP 5ml PBS + 1drop of A + 1drop of B( 1drop=50ul )
- Wash slides in 0.1% PBT for 5minX3.
- Incubate with ABC for 45-60min.
- Wash in 0.1% PBT for 5min X3.
- Wash in NTMT
- Color developing:
Blue Substrate: NBT/BCIP (Roche)
Red Substrate: Vector Red Alkaline Phosphatase Substrate (Vector SK-5100)
- Wash with ddH2O several times.
- Counter-stain with nuclear fast Red for 10-15 min or Hematoxylin for 1 min.
- Wash and dehydrate in air. Mount the slides with mounting medium (Vecta Mount. Vector H-5000).