By Rachel Stewart, PhD and Ryan Hamnett, PhD
Immunohistochemistry (IHC) involves using antibodies to selectively label proteins of interest in tissue samples. Below is our troubleshooting guide to help solve any issues that might be encountered with an IHC experiment.
| Potential Issue | Possible Solution |
|---|---|
| The epitope recognized by the primary antibody is not expressed, or expressed at very low levels in the tissue sample. | Ensure the antibody is compatible with the species of the tissue sample. Ensure the protein of interest is expressed in the tissue sample using western blotting, or existing protein and RNA databases. |
| The concentration of primary antibody was too low or the incubation time was too short. | Increase the concentration of the antibody and/or perform an antibody titration experiment to determine the optimal antibody dilution. Increase the incubation time. |
| The primary antibody cannot access its epitope in the tissue sample due to the fixation conditions (e.g. crosslinking in aldehyde fixation). | Perform antigen retrieval before immunostaining.Use alternative fixatives to maintain antigen availability. |
| The primary antibody does not recognize the target protein in its native (non-denatured) state/is only appropriate for immunoblotting. | Refer to the antibody’s data sheet to determine whether it is appropriate or has been validated for IHC. |
| If antigen retrieval was performed, the epitope recognized by the primary antibody was destroyed. | Optimize the antigen retrieval conditions, or consider not performing this step. |
| Antigen retrieval was ineffective. | Optimize the antigen retrieval conditions |
| Primary and secondary antibodies are incompatible | Ensure using a secondary antibody raised against the primary antibody host. |
| Antibodies are not working due to improper storage | Follow storage instructions provided. Typically, antibodies should be aliquoted in small volumes (e.g. 10 μl) and stored at –20°C or –80°C. Avoid repeated freeze-thaw cycles. |
| If using an enzyme-based detection method, the substrate and/or secondary antibody conjugate incubation conditions were suboptimal. | Increase the incubation time with the substrate.Ensure the substrate and conjugate are compatible.Ensure the substrate and conjugate are still active. |
| Potential Issue | Possible Solution |
|---|---|
| The primary or secondary antibody concentrations were too high. | Decrease the concentration of the antibody and/or perform an antibody titration experiment to determine the optimal antibody dilution. |
| Conditions of antibody incubations were suboptimal | Decrease the incubation time, particularly for room temperature incubations.Perform the incubation at 4°C.If performing free-floating or wholemount IHC, perform all incubations with shaking. |
| The blocking step was suboptimal | Ensure fresh reagents are used for blocking the tissue sample.Ensure using normal serum from the same species as the host of the secondary antibody.Increase the length of the blocking step, usually up to 1 hour.Increase the concentration of the blocking agent. |
| The secondary antibody bound non-specifically or bound to Fc receptors on cell surfaces | Include a control condition using only secondary antibody staining with no primary antibody incubation.Block the tissue sample using normal serum from the same species as the host of the secondary antibody.Use a different secondary antibody or one that specifically recognizes the Fab fragment of the primary antibody. |
| The secondary antibody cross-reacts with the tissue sample. | Use a secondary antibody that has been adsorbed with serum or immunoglobulins from the same species as the tissue sample. |
| The secondary antibody cross-reacts with an off-target primary or secondary antibody when multiplexing. | Ensure that primary antibodies are from distinct host species.Ensure secondary antibodies will not recognize other secondary antibodies, e.g. donkey anti-goat will recognize goat primary and secondary antibodies. |
| The primary antibody was raised in the same host species as the tissue sample. | Where possible, use a primary antibody raised in a different host species.Use a species-on-species detection kit. |
| The tissue sample was insufficiently washed. | Increase the length or number of washes. |
| The fixation method introduced autofluorescence (ex. formalin and paraformaldehyde produce autofluorescence in the green spectral range). | Use a fluorophore in the red or infrared spectral ranges. |
| The tissue sample dried out. | Perform all incubation steps in a humidified chamber. |
| Potential Issue | Possible Solution |
|---|---|
| Antigen retrieval methods are too harsh | Optimize antigen retrieval incubation length, or try a different retrieval method. |
| Tissue sections are not adhering to slide | Ensure tissue is dried on slide before freezing (for fresh-frozen tissue).Use freshly prepared (or purchased), properly charged slides. |
| Poor resolution | Cut thinner sections.Ensure adequate cryopreservation (for fixed-frozen tissue).Use fluorescence rather than chromogenic detection. |
| Tissue degraded due to slow or inadequate fixation | Fix tissue as soon as possible after fixation, or fix by perfusion.Increase fixation time. |
| Potential Issue | Possible Solution |
|---|---|
| Fixation method inappropriate for antigen | Optimize fixation conditions.Try an alternative fixative. |
| Fixation is introducing artifacts | Optimize fixation conditions.Try an alternative fixative. |
| Antigen has diffused due to a delay in fixation | Fix tissue as soon as possible after dissection.Try a cross-linking (aldehyde) fixative rather than an organic fixative. |