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scientificprotocols/protocol.md

Last active August 29, 2015 14:03
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Immunoperoxidase staining of culture cells
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protocol.md

Method:

  1. Grow cells on sterile coverglasses or chamber slides overnight.
  • Rinse briefly with PBS
  • Fix cells by incubation with one of the following methods:
    • 1% formalin in PBS for 10 minutes
    • 80% methanol in PBS for 10 minutes
    • Cold acetone for 5 minutes, air dry
    • 4% paraformahyde
  • Rinse three times with PBS
  • Incubate with 1% H2O2 in PBS for 10 minutes to quench endogenous peroxidase activity
  • Rinse with PBS twice
  • Incubate slides for 30 minutes in 10% blocking serum in PBS (suppresses non-specific binding of IgG)
  • Blot excess blocking serum
  • Incubate with primary antibody for 1 hour at room temperature or overnight at 4°C. Optimal antibody concentration is usually from 0.5-10ug/ml in TBS.
  • Wash three times with PBS
  • Incubate with biotin-conjugated secondary antibody for 30-45 minutes at room temperature. Again, optimal antibody concentration is usually from 0.5-10ug/ml in TBS
  • Wash three times with PBS
  • Incubate with streptavadin-HRP for 15 minutes. This step should be titrated as excess streptavadin-HRP can lead to high background
  • Wash with PBS three times
  • Incubate with freshly mixed DAB solution for 3-10 minutes. We highly suggest that you should observe the color developing under microscopy.
  • Rinse three times with distilled water
  • Counterstain with hematoxylin
  • Dehydrate with 70-100% alcohol and clear with xylene
  • Mount with coverslip

DOI

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