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Rabbit Anti-P-ERK in paraffin embedded sections

  1. Deparaffinize 20minX2. Rehydrate slides to ddH2O.
    Wash in ddH2O 3min X2.
  2. Antigen retrieval:
    Set water bath to 95-99°C and prewarm 200ml ddH2O.
    Add 1.9 ml Antigen Unmasking Solution (Vector) to 200ml ddH2O.
    Leave in water bath for 20 min.
  3. Wash in PBS 3min X2.
  4. Block endogenous peroxidase with 3% hydrogen peroxide in PBS) for 15 min.
  5. Wash in PBST 2min X3.
  6. Block non-specific proteins with 10%  normal goat serum for 30-60min at RT.
  7. Incubate with P-ERKAb 1:50-100 (Cell Signaling Cat#9101). Incubate O/N  at 4°C.
  8. Wash slides with PBST 5min X4.
  9. Incubate with 1:200 Biotinylated goat anti-rabbit IgG (Vector) 1 hour at RT.
  10. Make up ABC (Vector):
    ABC 2.5ml PBS+ 1drop of A + 1drop of B( 1drop=50ul )
  11. Wash slides in PBST for 5minX4.
  12. Incubate with ABC for 45-60 min.
  13. Wash in PBST for 5min X4.
  14. Wash in 0.1MTris pH 7.5 and 0.3M NaCl 5minX2.
  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. Mount the slides with mounting medium.

 

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