Immunology

scientificprotocols authored about 3 years ago

Method:

  1. Grow cells on sterile coverglasses or chamber slides overnight.
  2. Rinse briefly with PBS
  3. 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
  4. Rinse three times with PBS
  5. Incubate with 1% H2O2 in PBS for 10 minutes to quench endogenous peroxidase activity
  6. Rinse with PBS twice
  7. Incubate slides for 30 minutes in 10% blocking serum in PBS (suppresses non-specific binding of IgG)
  8. Blot excess blocking serum
  9. 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.
  10. Wash three times with PBS
  11. 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
  12. Wash three times with PBS
  13. Incubate with streptavadin-HRP for 15 minutes. This step should be titrated as excess streptavadin-HRP can lead to high background
  14. Wash with PBS three times
  15. Incubate with freshly mixed DAB solution for 3-10 minutes. We highly suggest that you should observe the color developing under microscopy.
  16. Rinse three times with distilled water
  17. Counterstain with hematoxylin
  18. Dehydrate with 70-100% alcohol and clear with xylene
  19. Mount with coverslip

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