We are dedicated to helping our customers achieve exceptional results. While the information below can never be an exclusive solution to every problem you may encounter; it is our hope that you will find the information useful and even beneficial in troubleshooting any problems you may have. Of course, if you find you are still having problems you may submit them to us using this IHC-P Troubleshooting Form.




Lack of staining

Lack of antigen

Check protein expression by in situ hybridization.

Improper storage of antibodies

Follow storage instructions on the datasheet. Aliquot antibodies in a sufficent volume 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

Check antibodies independently on a dot blot.

Inadequate tissue fixation

Increase or decrease fixation incubation, and try to change the fixative.

Over fixation

Reduce the duration of the immersion or post-fixation steps and be sure to include the Antigen Retrival step.

Antigen destroyed prior to staining

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 embedding or fixation

Embed tissue at 58 °C or below. Try restoring immunoreactivity through various antigen retrieval techniques.

Incomplete deparaffization

Use fresh xylene and increase xylene incubation time.

Insufficient antibody incubation

Incubate primary antibody overnight at 4°C


Ensure to proceed with all steps in the dark.

Antigen retrieval was ineffective

Try to change the incubation time and also change solutions.

Antibodies are not compatible

Make sure you use a secondary antibody that was raised against the primary antibody species. Make sure that the isotypes of the primary and secondary are compatible.

High background

Hydrophobic interactions of the antibody and proteins in the tissue

Lower the salt concentration of the antibody stain buffer.

Tissue sections have dried out

Examine the tissue; sections with higher background staining at the edges than towards the center are often dried out. Prevent tissue sections drying out by keeping them in a humidified chamber.

Non-specific binding of primary antibody

Use the blocking step just prior to primary antibody incubation.

Non-specific binding of secondary antibody

Use an antibody that has cross-reactive IgG species removed (pre absorbed against sample species).

Ionic interactions

Ensure to change buffers.

Antibody is binding to Fc receptors on the target's cell surface

To block open binding sites, use 10% serum of the host secondary antibody.

Incubation temperature may be too high

Be sure to incubate at a temperature of 4°C.

Tissue inadequately washed

Include washing steps with fresh PBS/TBS.

Endogenous peroxidase

Use a peroxidase quenching buffer in blocking step before primary antibody.

Permeablization of membrane

Be sure to remove detergents from buffers.

Fixative was too strong

The epitope was modified, so be sure to change the antigen retrieval method.

Non-specific staining 

Antibody incubation too long

Lessen the primary antibody incubation time.

Endogenous peroxidase

Use a peroxidase quenching buffer in the blocking step before the primary antibody.

Sections have dried-out

Ensure to keep slides in a humidity chamber.

Delay in fixation

Be sure to fix the tissue immediately.

Insufficient washing/blocking

Increase the washing time and number of washes.


Primary antibodies too concentrated

Dilute primary antibody solution. Perform a titration to determine optimal antibody dilution.

No or weak staining

Tissues have dried out

Cover the tissues in liquid at all times during the experiment.