Appropriate controls are crucial for the evaluation of staining specificity. Negative controls are essential to identify false positive staining.
SYSY recommends performing the following staining controls to support the specificity of your stainings.
Slide without Primary Antibody and Secondary System
When using chromogenic detection in IHC, endogenous peroxidases or alkaline phosphatases can produce false positive staining when not blocked appropriately. Omitting the primary antibody and the secondary system in the staining protocol will reveal endogenous enzyme activity. Use 3% H2O2 to block endogenous peroxidases. Endogenous alkaline phosphatases can be blocked with 1 mM Levamisole.
Other false positive signals inheritent to the tissue itself that can be easily confounded with the DAB signal due to yellow-brown color are: hemosiderine (e.g. liver, spleen, and bone marrow), bilirubin (liver) lipofuscin (e.g. nerve cells, heart, and liver), and melanin in corneal endothelial cells (brown eyes) and human skin cells.
In fluorescent IHC-P, tissue autofluorescence may be confused with specific signals or unspecific background.