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HOME > Protocols > Histology > Staining protocols > Protocol for Immunoperoxidase staining of frozen tissue sections

Protocol for immunoperoxidase staining of frozen tissue sections

  1. Clean slides with 95% ethanol
  2. Treat with a solution containing 0.3% gelatin and 0.05% chromium poatassium sulfate
  3. Cut 6 to 8 micron thick cryostat sections of tissue block
  4. Allow frozen sections to come to room temperature
  5. Fix slides in cold acetone (-20oC) for 10 minutes
  6. Rinse three times with PBS
  7. Incubate for 10 minutes with 0.1% H2O2 in PBS to quench endogenous peroxidase activity
  8. Rinse once with PBS
  9. Incubate slides for 20 minutes in 10% goat serum in PBS (suppresses non-specific binding of IgG)
  10. Wash with PBS
  11. 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
  12. Wash three times with PBS
  13. Incubate with biotin-conjugated secondary antibody for 45 minutes. Again, optimal antibody concentration is usually from 1-10ug/ml in PBS-BSA
  14. Wash three times with PBS
  15. Incubate with streptavadin-HRP for 15 minutes. This step should be titrated as excess streptavadin-HRP can lead to high background
  16. Wash with 0.5% triton x-100 in PBS for 30 seconds
  17. 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
  18. Rinse with distilled water
  19. Counterstain with hematoxylin
  20. Dehydrate with alcholos and xylene
  21. 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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