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Avidin-Biotin Complex (ABC) Method

The following is a general ABC method, including the steps that are involved with the correct performance of an ABC method. The order of the steps can be changed and some may be omitted. This is a common procedure that is used on automated stainers and for hand staining using the ABC method and no polymers. The manufacturers of the automated systems usually give recommendations for solutions and antibodies and how to use them. A technician needs to understand what is going on when using an automatic stainer and how to identify errors and fix them.

Paraffin-Embedded Tissue

  1. Begin deparaffinization with 100% xylene/substitute
  2. 100% alcohol/substitute
  3. 95% alcohol/substitute
  4. Distilled water
  5. Antigen retrieval (optional)
  6. Rinse in distilled water
  7. Enzyme @ 37 C (optional)
  8. Distilled water
  9. 3.0% hydrogen peroxide (H2O2) diluted in methanol (to prepare, add 10 ml 30% H2O2 to 90 mL methanol) Note: This step can also be done after the primary antibody incubation.
  10. Rinse in the solution used to dillute the H2O2
  11. Buffer rinse to prepare for the staining
  12. Block
  13. Primary antibody
  14. Buffer rinse
  15. Link (optional)
  16. Buffer rinse
  17. Label (ABC)
  18. Buffer rinse
  19. Substrate
  20. Running tap water or recommended solution
  21. Mayer's hematoxylin (optional)
  22. Dehydrate in graded alcohols, clear in xylene, and mount with resinous media if not IF type