20-Step IHC-Paraffin Protocol Example

  1. Mount and section samples 
  2. Heat sections on the specimen slide to improve adherence 
  3. Remove paraffin and rehydrate the tissue 
  4. (Optional) Perform heat induced or protease induced epitope retrieval 
  5. Wash 
  6. Block endogenous peroxidases/phosphatases (for enzymatic labels) and biotin (for biotin/avidin detection systems) 
  7. Wash 1x in phosphate buffered saline (PBS) 
    Use Tris-buffered saline (TBS) instead of PBS for detecting phosphoproteins or when using alkaline phosphatase (AP) conjugated antibodies 
  8. Block non-specific binding sites 
  9. Wash 3x in PBS or TBS 
  10. Incubate with primary antibody 
  11. Wash 3x in PBS or TBS
  12. (Optional) Incubate with secondary antibody 
  13. Wash 3x in PBS or TBS 
  14. (Optional) Incubate with amplification reagent 
  15. Wash 3x in PBS or TBS
  16. Incubate with DAB or other substrate solution (for enzymatic labels only) 
  17. Wash in ddH2
  18. Counterstain 
  19. Dehydrate tissue sections (for organic mounting media only)
  20. Mount coverslip