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Further Enhancements of ABC Protocols

Although standardised protocols are important to develop and adhere to wherever possible, some antigens may need a little extra help. Variations should always be validated to ensure results are not altered, so it is important to run appropriate controls.

For the most part, the standard protocols provided by kit manufacturers are best followed and optimization is usually achieved by adjusting the concentration of the primary antibody - recomended for reproducibility. However, sometimes the required concentration of primary antibody can make them costly to use, so increasing the sensitivity of the detection system to allow for further dilution of the primary can be appealing. 

Biotinylated Anti-Avidin

Biotinylated anti-avidin can be a useful linker tool - not only can it detect avidin via the antibody end, but also conjugate via the avidin-biotin interaction. Here it is used to build a second layer of ABC reagent. 

“Enhanced” ABC Immunohistochemistry Method

(A method developed at Morbid Anatomy Dept., Royal London Hospital provided to Vector Laboratories)

  1. De-wax sections and take to alcohol
  2. Block endogenous peroxidase                                        
  3. Wash in tap water                                                               2 mins.
  4. Antigen unmasking (if required)
  5. Wash in tap water                                                               2 mins.
  6. Transfer to TBS pH 7.6                                                      3 mins.
  7. Add 20% normal horse serum block                                  3 mins.
  8. Tip off and apply primary antibody                                                 
  9. Wash in TBS (x2)                                                         
  10. Apply biotinylated secondary antibody (1:100)                 20 mins.
  11. Wash in TBS (x2)              
  12. Apply VECTASTAIN® Elite ABC complex                      20 mins.
  13. Wash in TBS (x2)                                                                   
  14. Apply Biotinylated Anti-Avidin D: BA-0300 (1:150)        10 mins.
  15. Wash in TBS (x2)                                                                2 mins.
  16. Apply VECTASTAIN® Elite ABC complex                     10 mins.
  17. Wash in TBS (x2)                                                                 
  18. Apply substrate solution                                                       5-10 mins.
  19. Wash in TBS (x2)                                                             
  20. Counterstain & differentiate
  21. Dehydrate, clear & mount