Troubleshooting - High background

Immunohistochemistry (IHC) troubleshooting for no staining, high background and non-specific staining.

Problems Possible reasons Solutions
High background Antibody incubation time was too long Decrease the antibody incubation time
Polyclonal primary antibody Try a monoclonal primary antibody
Serum blocking was absent or insufficient Increase the blocking time
High antibody concentration Optimize the antibody concentration
Presence of endogenous peroxidase Block endogenous peroxidase activity properly
Insufficient PBS washing Wash with PBS for long enough and for enough times to eliminate fixatives

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