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.
- Following incubation with a blocking serum, incubate with avidin for 15 minutes.
- Briefly immerse in wash solution.
- Incubate with biotin solution for 15 minutes.
- Briefly immerse in in wash solution to eliminate residual biotin.
- 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.
- Add 150 ul of avidin solution per ml of serum block.
- Following serum block, briefly rinse tissue in wash solution.
- Add 150 ul of biotin solution per ml of primary antibody solution, and proceed with primary antibody incubation.