Troubleshooting in IHC


Possible causes





Poor or ​No staining


Primary and secondary antibodies are not compatible

·         Use a secondary antibody that was raised against the species of the primary antibody - e.g. if the primary antibody was raised in mouse, use a secondary anti-mouse.

·         Ensure that the isotypes of the primary and secondary are compatible.





Low abundance in protein of interest

·         Use signal amplification to increase the signal -e.g. a biotin-conjugated secondary antibody.





Formalin and paraformaldehyde fixatives may mask the epitope

·         Perform an antigen retrieval treatment to unmask the epitope – e.g. heat mediated with pH 6 citrate buffer, enzymatic, etc.

·         Decrease fixation time.




High background


Primary antibody concentration may be too high

·         Dilute the antibody to its optimal concentration.





Secondary antibody may be binding non-specifically

·         Ensure that you use a secondary antibody raised in a different species to your sample.

·         Try a secondary antibody that has been pre-adsorbed against the lg of the species of your samples.

·         Run a control without primary antibody. 





Endogenous enzymes are active, if using enzyme-conjugated antibody, and may give false positives

·         Block them with inhibitors prior to immunostaining:

o    for peroxidase (HRP conjugated antibodies), use Hydrogen Peroxide (0.3% v/v).

o    for biotin-based IHC detection, use Avidin/Biotin blocking reagents.

o    for alkaline phosphatase (AP conjugated antibodies), use Levamisol (2mM).





Overstained Tissue

Primary and/or secondary antibody concentration is too high

·         Determine the optimal primary antibody concentration by staining adjacent sections.





Nonspecific binding of primary and/or secondary antibodies to tissues

·         Treat tissues to minimize or block nonspecific binding.




Altered Tissue Morphology and Microscopy         


Antigen retrieval technique was too harsh

·         Try a different treatment or optimize method to make conditions less harsh.





Frozen sections detach from slide

·         Increase fixation time - use freshly prepared slides.





No resolution of sections at the single-cell layer level

·         Prepare thinner sections.





Tissue appears damaged 

·         Increase fixation time - use smaller pieces of tissue. Fixative must have access to the interior of the tissue.