Troubleshooting Guide: Immunohistochemistry
This protocol is intended as a guide only.
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Easy to follow protocols and troubleshooting tips.
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. | Follow storage instructions on the datasheet. In general, aliquot antibodies into smaller volumes sufficient to make a working solution for a single experiment. Store aliquots in a manual defrost freezer (-20 to -70 °C) and avoid repeated freeze-thaw cycles. |
| Inactive primary or secondary antibodies. | Test reporter system independently to assess reagent viability. Replace with a new batch of antibodies if needed. |
| Inadequate tissue fixation. | Try increasing the fixation time or try a different fixative. |
| Tissue overfixation. | Reduce the duration of the immersion or post-fixation steps. If immersion fixation cannot be avoided (for example, collection of postmortem tissues or biopsies in pathology lab), antigens may be unmasked by treatment with antigen retrieval reagents. |
| Incompatible secondary and primary antibodies. | Use a secondary antibody that will interact with the primary antibody. For example, if the primary antibody was raised in rabbits, use an anti-rabbit secondary antibody. |
| Inactive secondary reagents. | Replace with new reagents. |
| Antigen was destroyed before incubation with the primary antibody. | If quenching of endogenous peroxidase was done prior to the addition of primary antibodies, block peroxidase after incubation with the primary antibody. |
| Epitope altered during fixation or embedding procedure. | Try restoring immunoreactivity through various antigen retrieval techniques. Embed tissue at 58 °C or below. |
| Antigen retrieval was ineffective. | Increase the time of treatment or change the treatment solution. |
| Reagents omitted or used in wrong order. | Repeat staining and confirm that correct reagents are used and are added in the correct order. |
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 |
|---|---|
| High concentration of primary and or secondary antibodies. | Titer antibody to determine optimal concentration needed to promote a specific reaction of the primary and the secondary antibodies. |
| Hydrophobic interactions of the antibody and proteins in the tissue. | Lower the ionic strength of the antibody diluent (particularly monoclonal antibodies respond well to reducing the salt concentration). |
| Non-specific binding of primary and/or secondary reagents to tissues. | Use blocking step just prior to primary antibody incubation (we use 1% bovine serum albumin with 10% normal donkey serum). Non-fat dry milk is another option. |
| Non-specific binding of secondary antibody. | Use an antibody that has cross-reactive IgG species removed (absorbed against sample species). |
| Tissue dried out. | Avoid letting the tissue dry during the staining procedure. |
| Reagents sticking to old or poorly prepared slides. | Start over with freshly prepared or purchased slides. |
| Background due to ionic interactions. | Increase the ionic strength of the diluent buffer. |
| 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 |
Problem: Cell/Tissue Morphology is Destroyed
| Possible Source | Test or Action |
|---|---|
| Antigen retrieval methods are too harsh. | Empirically determine the conditions that preserve tissue morphology while restoring the immunoreactivity of the antigen. |
| Tissue sections falling off slide. (more common with frozen sections) | Increase fixation time. Empirically determine an additional or alternative fixative. Use freshly prepared, adequately charged slides. |
| Tissue section appears torn or folded. Air bubbles under section. | Re-cut sections using a sharp blade, or ignore damaged areas when analyzing the results. |
| Poor resolution of tissue morphology | Cut thinner tissue sections. Ice crystals may have destroyed morphology of frozen sections. |
| Underfixation has physically damaged the tissue or cells during the staining process | Increase fixation time and/or add a post-fixation step. Increase the fixative/tissue ratio. Cut smaller pieces of tissue for more thorough immersion fixation. |
| Autolysis of tissue leading to staining of necrotic debris. | Increase the fixation time, ratio. Consider using cross-linking fixative. |
Problem: Staining is Inappropriate
| Possible Source | Test or Action |
|---|---|
| Fixation method is inappropriate for the antigen. | Try a different fixative or increase the fixation time. |
| Antigen retrieval may be inappropriate for this antigen or tissue. | Try different antigen retrieval conditions. |
| Electrostatic charge of the antigen has been altered. | Try adjusting the pH or cation concentration of the antibody diluent. |
| Delay in fixation caused diffusion of the antigen. | Fix tissue promptly. Try a cross-linking fixative rather than organic (alcohol) fixative. |