Avidin-Biotin Blocking Solution


Biotin-avidin labeling is  commonly used as a method for improving staining intensity in immunohistchemical techniqes. However, a high non-specific background is a common problem with this approach, since many tissues contain abundant quantities of biotin-binding proteins or  nonspecific binding sites. One approach to reduce non-specific background is to pre-treat tissue sections with  solutions of avidin followed by biotin. Tissues from kidney, liver, and spleen usually contain a high level of endogenous biotin, requiring a biotin-avidin block.

In cases where it is not clear if an biotin-avidin block is required, tissues can be tested using a simple procedure. Endogenous peroxidase is first neutralized through a hydrogen peroxide blocking step, and then tissue is  incubation with strepavidin-HRP followed by visualization with DAB.


Avidin in Phosphate Buffered Saline

  • Prepare 0.001% avidin (10 U/mg or greater) in  phosphate buffered saline (PBS).
  • Store at 4oC.

Biotin in Phosphate Buffered Saline

  • Prepare 0.001% biotin  in  phosphate buffered saline (PBS).
  • Store at 4oC.


  1. Following incubation with a blocking serum, incubate with avidin for 15 minutes.
  2. Briefly immerse in wash solution.
  3. Incubate with biotin solution for 15 minutes.
  4. Briefly immerse in in wash solution to eliminate residual biotin.
  5. Proceed with addition of primary antibody for immunohistochemistry.

Rapid Avidin-Biotin Blocking Procedure

In many cases, a rapid avidin-biotin blocking procedure can be used in place of the stepwise blocking procedure. This approach combines avidin-biotin blocking with the serum and primary antibody incubation steps. It should not be used if the  primar antibody is biotinylated.

  1. Add 150 ul of avidin solution per ml of  serum block.
  2. Following serum block, briefly rinse tissue in wash solution.
  3. Add 150 ul of  biotin solution per ml of primary antibody solution, and proceed with primary antibody incubation.

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