Immunohistochemistry (IHC) Troubleshooting


ProSci Inc has answers for all your Immunohistochemistry(IHC) troubleshooting needs. Consult our Immunohistochemistry protocol and these immunohistochemistry tips for all stages of IHC protocol.

High Background or Non-Specific IHC Staining

For IHC troubleshooting high back ground or non-specific IHC staining consider following:

Endogenous Enzyme

Block endogenous enzyme (peroxidase) activities using 3% hydrogen peroxide prior to incubation of primary antibody if HRP-based detection system is being used.

Endogenous Biotin

Block endogenous biotin activity (i.e. kidney and liver tissues) using the avidin/biotin blocking reagent prior to incubation of primary antibody, after the normal blocking procedure.


Increase the blocking incubation duration and consult Prosci technical support for the recommended blocking reagent.

Antibody Dilution/Diluent

Run a serial dilution test to determine the optimal dilution. Consult Prosci product datasheet for the recommended dilution. Higher dilution of primary antibody can reduce the non-specific binding.

Secondary Antibody Cross Reactivity

Run a secondary control without primary antibody. If positive, change your secondary or use a secondary antibody that has been pre-absorbed against the Ig of the species of your samples. Alternatively, reduce the concentration of the biotinylated secondary antibody.

Primary Antibody (Mouse on Mouse tissue)

Use a primary antibody raised against a different species than your tissue. Or use a biotinylated primary antibody and a conjugated streptavidin for the detection system.

Amplification/Detection System

Too much amplification or substrate can lead to non-specific staining or high background. Reduce amplification/substrate incubation time and dilute the secondary antibody or amplification kit.


Wash extensively in PBS between all steps.

Incubation Temperature

Incubate slides overnight at 4°C.

Slides dried out

Keep slides at high humidity and avoid sections being dried out.

Weak or No Staining

Tissue Overfixation

Reduce the duration of post-fixation. If the tissue has already been over-fixed, perform an appropriate or recommended antigen retrieval procedure.

Inadequate Deparaffinization

Deparaffinize sections longer or use fresh xylene

Antigen Retrieval

Target protein staining may require an optimized unmasking buffer and protocol since epitope may change during fixation or embedded procedure. Consult Prosci technical support for the recommended solution. Always prepare fresh 1X buffer daily.

Antibody Preparation

Improper storage or handling can lead to inactive antibody or antibody aggregation. Aliquot antibodies into smaller volumes and store in freezer (-20 to -70 C) and avoid repeated freeze and thaw cycles. Or store antibodies according to manufacture's instructions.

Antibody Dilution/Diluent

Run a serial dilution test to determine the optimal dilution. Consult Prosci product datasheet or technical support for the recommended dilution. Increase the concentration of primary and/or secondary antibodies.

Incubation Duration

Incubate overnight at 4°C is highly recommended.

Detection System

Verify the expiration date of the detection reagent prior to use. Use biotin-Streptavidin system to maximize the signal.

Using Control

Positive Control

Validate the staining in the sample is valid and verify that the primary/secondary antibody or protocol is working properly. Check the antibody datasheet and consult Prosci technical support for the high-expressing suitable positive control.

No Primary Control

The primary antibody is not added to the sample and use antibody diluent instead. This will check if any non-specific binding or false positive result may be due to non-specific binding of the secondary antibody.

Isotype Control

Use isotype control on your sample instead of the specific primary antibody. The isotype control is an antibody of the same isotype, clonality, conjugate, and host species as the primary antibody that is raised against a molecule that is non-existent in the sample you are using. The concentration of the isotype control antibody should be as same as the primary antibody. This will check the level of background in your sample.