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HOME > Protocols > Cell Biology > Cell Culture Protocols > Protocol for Immunoperoxidase staining of tissue culture cells

Protocol for Immunoperoxidase staining of tissue culture cells

  1. Grow cells on sterile glass cover slips or slides overnight in 37oC/5% CO2 incubator
  2. Rinse briefly with PBS
  3. Fix cells by incubation with 1% formalin in PBS for 10 minutes
  4. Rinse three times with PBS
  5. Incubate for 10 minutes with 0.1% H2O2 in PBS to quench endogenous peroxidase activity
  6. Rinse once with PBS
  7. Incubate slides for 20 minutes in 10% goat serum in PBS (suppresses non-specific binding of IgG)
  8. Wash with PBS
  9. Incubate with primary antibody for 1 hour at room temperature or overnight at 4oC. Optimal antibody concentration is usually from 1-10ug/ml in PBS-BSA
  10. Wash three times with PBS
  11. Incubate with biotin-conjugated secondary antibody for 45 minutes. Again, optimal antibody concentration is usually from 1-10ug/ml in PBS-BSA
  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 0.5% triton x-100 in PBS for 30 seconds
  15. Incubate with fresh DAB solution for 5 minutes. This step can be lengthened if additional sensitivity is necessary. It is a good idea to do a quick spot check here
  16. Rinse with distilled water
  17. Counterstain with hematoxylin
  18. Dehydrate with alcholos and xylene
  19. Mount permanent coverslip

Note: Incubations are to be at room temperature or a 4oC humidified chamber. Antibody concentrations here may not hold for all sources. These concentrations should be titrated for each source.


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