Troubleshooting Guide: Immunohistochemistry

Problem: Lack of Staining

Possible Source	Test or Action
Lack of antigen	Check protein expression by in situ hybridization (in some rare cases translation may be blocked even though mRNA is detected)
Antibodies do not work due to improper storage	Aliquot antibodies into smaller volumes sufficient to make a working solution for a single experiment. Store aliquots in a freezer (-20 to -70°C) and avoid repeated freeze-thaw cycles
Inactive primary antibodies	Replace with a new batch of antibodies
Inadequate tissue fixation	Try different fixative
Tissue overfixation	Reduce duration of postfixation or fix tissues at 4° C. If postfixation can not be avoided (for example, collection of postmortem tissues in pathology lab) antigens may be unmasked by treatment with antigen-retrieval reagents
Incompatible secondary and primary antibodies	Use secondary antibody that will interact with the primary antibody. For example, if primary antibodies were raised in rabbits, use anti-rabbit secondary but not anti-mouse or anti-goat
Inactive secondary reagents	Replace with new reagents
Antigen was destroyed before incubation with primary antibody	Quenching of endogenous peroxidase was done prior to the addition of primary antibodies. Block peroxidase after incubation with primary antibody

Problem: Overstaining

Possible Source	Test or Action
High concentration of primary and/or secondary antibodies and/or reagent	Determine optimal concentration for each component of immunohistochemical reaction: primary antibodies; secondary antibodies; enzymes catalyzing formation of color precipitates
Long incubation time	Determine optimal incubation time for each component of immunohistochemical reaction: primary antibodies; secondary antibodies; enzymes; chromogenic substrates
Non-specific binding of primary and/or secondary reagents to tissues	Treat tissues to reduce or block non-specific binding of immunohistochemical components to tissues, for example, by Avidin-Biotin Blocking Reagents

Problem: High Background

Possible Source	Test or Action
Tissues have endogenous molecules which are also used in incubation mixtures, for example, the presence of peroxidase in blood cells or the presence of autofluorescent pigment lipofuscin in neuronal cells	Incubate with normal serum obtained from species other than from the species that were the source of the primary antibody Block endogenous component. Endogenous peroxidase may be blocked by incubating tissues for 30 minutes at room temperature with 0.3% H2O2 in methanol, or switch to protocols utilizing substances which are not present in tissues For autofluorescence: after finishing IHC staining, treat tissues with 1% Sudan Black in 70% alcohol. If residual autofluorescence still obscures specific labeling, use non-fluorescent enzymatic protocols with chromogenic substrates (for instance DAB or AEC) or employ immunogold-silver staining