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Immunostaining in paraffin embedded sections

ABC-AP (Alkaline Phosphatase)

1. Deparaffinize 20minX2. Rehydrate slides to ddH₂O.
   (100% ETOH 2min. 95% ETOH 2min. ddH₂O 2min X2).
2. Wash in 0.1% PBT 3min X2.
3. Block non-specific proteins with 10% goat serum in 0.5% PBT (PBS+0.5% Tween 20) for 30-60 min at RT.
4. Incubate with mouse or rabbit antibody in 5% goat serum O/N at 4°C.
5. Wash slides with 0.1% PBT 5min X3.
6. Incubate with 1:200 Biotinylated goat anti-mouse or rabbit IgG (Vector)in 0.1% PBT for 1hour at RT.
7. Make up ABC-AP (Vector) 30min before incubation.
   ABC-AP 5ml PBS + 1drop of A + 1drop of B( 1drop=50ul )
8. Wash slides in 0.1% PBT for 5minX3.
9. Incubate with ABC for 45-60min.
10. Wash in 0.1% PBT for 5min X3.
11. Wash in NTMT
12. Color developing:
    
    Blue Substrate: NBT/BCIP (Roche)
    MTNT 5ml
    NBT 25ul
    BCIP 19ul
    
    Red Substrate: Vector Red Alkaline Phosphatase Substrate (Vector SK-5100)
    
    
13. Wash with ddH₂O several times.
14. Counter-stain with nuclear fast Red for 10-15 min or Hematoxylin for 1 min.
15. Wash and dehydrate in air. Mount the slides with mounting medium (Vecta Mount. Vector H-5000).

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