IHC Troubleshooting: No Staining

The good immunohistochemical staining is a foundation of correct judgment for staining, since there are many steps and links through the whole immunohistochemistry process, each step and links are likely to affect the final result of the staining, so to do an immunohistochemistry quality slice is not a very easy thing.

After staining, sections do not see any positive signal, that is, no staining, This is a relatively common phenomenon in immunohistochemistry, there are many reasons for this phenomenon, introduce as follow:

  Possible reasons Solutions
1 Wrong primary antibody Choose an antigen-specific antibody
2 Inappropriate concentration(usually too low) of primary or secondary antibody Optimize the concentration
3 The activities of primary/secondary antibody lost. Check the antibody storage condition and run a positive control
4 Inappropriate pH of antibody diluents Make sure antibody works properly
5 Insufficient deparaffinization Deparaffinize sections longer
6 Insufficient permeabilization Add an appropriate permeabilizing agent
7 Insufficient antigen retrieval Follow the antigen retrieval protocol
8 The secondary antibody was not stored in the dark Wash with PBS for long enoug and for enough times to eliminate fixatives