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Rat Anti-PECAM-1(CD31) in Paraffin Sections

  1. Deparaffinize 20minX2. Rehydrate slides to ddH2O
    Wash in ddH2O 3min X2.
  2. Block endogenous peroxidase with 3% hydrogen peroxide in PBS for 15 min.
  3. Wash in PBS 3min X2.
  4. 0.1% Trypsin incubate for 10 min at 37°C.
    2.5% Trypsin 1ml + 24ml 0.1M Tris pH 7.6
  5. Wash in PBST 2min X3.
  6. Block non-specific proteins with 2%  normal rabbit serum with 2% Triton X-100 in PBS for 2 hours at RT.
  7. Incubate with Rat Anti-PECAM Ab 1ug/ml (1:500 Pharmingen 01951D)in blocking serum O/N at 4°C.
  8. Wash slides with PBST 5min X6.
  9. Incubate with 1:200 Biotinylated rabbit anti rat IgG (Vector BA-4001) 1 hour at RT.
  10. Make up ABC-HRP  (Vector PK-6100) ABC 2.5ml PBS+ 1drop of A + 1drop of B( 1drop=50ul )
    (Make up ABC-HRP 30min before use)
  11. Wash slides in PBST for 5minX6.
  12. Incubate with ABC for 45 min.
  13. Wash in PBST for 5min X6.
  14. Wash in 0.1MTris pH 7.5 and 0.3M NaCl.
  15. Color developing:
    A. DAB: One set tablets (Sigma) to 5ml ddH2O.
  16. Add DAB to slides for 2-10min.
  17. Wash with ddH2O several times.
  18. Counter-stain with Hematoxylin for 1 min.
  19. Wash and dehydrate in air. Mount the slides with mounting medium.

 

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