Avidin-Biotin Complex (ABC) Method

The lab utilizes the ABC method on all of its IHC staining protocols. Staining results are significantly improved by the pretreatment of the sections in an antigen retrieval soloution (DakoCytomation Target Retrieval Solution) under controlled heat and pressure using a Decloaking Chamber (Biocare Medical). The Target Retrieval Solution acts to break protein cross links formed by fixation in 10% Neutral Buffered Formalin, thus uncovering hidden antigenic sites.

Background staining is reduced by the application of a protein block (DakoCytomation Serum-Free Protein Block) directly to the sections to prevent non-specific background staining typically seen when performing IHC on animal tissue. Endogenous peroxidase activity, which is found in many tissues, is also blocked by the pretreatment of the sections with hydrogen peroxide prior to the application of the primary antibody.

The following is the immunohistochemistry protocol utilized by the Histology / IHC lab:

  1. Deparaffinize in xylene and hydrate sections to distilled water.
  2. Pretreat sections in Target Retrieval Solution.
    1. Add 500ml of distilled water to the decloaking chamber.
    2. Place slides in plastic coplin jar containing Target Retrieval Solution.
    3. Enclose slides and affix lid of decloaking chamber.
    4. Press start (slides are heated to 125 degrees Celsius).
    5. Slides will cool to 90 degrees Celsius for 10 seconds.
    6. Remove lid and allow slides to cool for 10 minutes.
  3. Wash buffer for 5 minutes.
  4. Rinse in distilled water.
  5. 3% Hydrogen Peroxide for 5 minutes.
  6. Rinse in distilled water.
  7. Rinse in wash buffer.
  8. Serum-free protein block for 10 minutes.
  9. Blow off protein block.
  10. Incubate with primary antibody for 30 minutes.
  11. Rinse in wash buffer.
  12. Incubate with biotinylated secondary antibody for 30 minutes.
  13. Rinse in wash buffer.
  14. Incubate with avidin-biotin complex for 30 minutes.
  15. Rinse in wash buffer.
  16. Rinse in water.
  17. Incubate with chromagen (DAB) for 5 minutes.
  18. Counterstain with hematoxylin for 2 minutes.
  19. Dehydrate, clear in xylene and coverslip.