Troubleshooting - No staining

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

Problems Possible reasons Solutions
No staining Wrong primary antibody Choose an antigen-specific antibody
Inappropriate concentration (usually too low) of primary or secondary antibody Optimize the concentration
The activities of primary/secondary antibody lost. Check the antibody storage condition and run a positive control
Inappropriate pH of antibody diluents Make sure antibody works properly
Insufficient deparaffinization Deparaffinize sections longer
Insufficient permeabilization Add an appropriate permeabilizing agent
Insufficient antigen retrieval Follow the antigen retrieval protocol
The secondary antibody was not stored in the dark Biotinylated secondary antibody should be stored in the dark.

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