Immunohistochemistry Troubleshooting Guide

Technically, IHC and ICC are relatively simple and straightforward experimental methods. However, there are many variables which must be identified and optimized for each individual IHC/ICC study. Optimization of IHC/ICC may also require troubleshooting a variety of factors. The tables below highlight common IHC/ICC issues and provide appropriate experimental actions. For further assistance, please contact our technical service department.

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 cannot 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