IHC Troubleshooting

Potential cause	Suggested test or solution
The primary/secondary antibody lost its activity.	Use a new lot of antibody. Improper storage of antibody. Follow manufacturer’s instruction. Normally, prepare single-use aliquots and store at -20°C. Extensive thaw-freezing cycles have damaged the antibody.
Conditions of antibody are not optimized.	Titrate the antibody concentration to optimize best working conditions. Incubate the primary antibody at room temperature or at 4°C overnight.
Protein of interest is not expressed in used tissue.	Run a positive control
Protein of interest is low expressed in used cells.	Use signal amplification when visualizing.
Damaged epitope.	Change to another antigen retrieval buffer/technique for paraffin-embedded samples.
Antibody is not suitable for this application.	Check validation data of manufacturer.

Potential cause	Suggested solution
Too high primary/secondary antibody concentration.	Titration of antibodies to determine optimal working concentration.
Non-specific binding of primary/ secondary antibodies.	Prolong blocking step and increase concentration of blocking solution. Run positive and negative controls.
Non-specific binding of secondary antibodies.	Run control with secondary antibody only. Change to a cross-adsorbed secondary antibody or a fragment antibody.
The sample is poorly washed.	Repeat or prolong washing step.
The antibody incubation temperature/time is not suitable.	Optimize conditions.
Damaged epitope.	Change to another antigen retrieval buffer/technique for paraffin-embedded samples.

Potential cause	Suggested solution
Harsh antigen retrieval conditions	Optimize buffers, temperature, pH, incubation time, concentration.
Unclear tissue structure	Optimize thickness of tissue slides. Cut new sections.
Tissue is not adhesive to glass slide	Optimize fixation. Decrease heating time or temperature during HIER.
Physically damaged cell shape.	Under-fixation. Change fixative or fixation time.