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IHC Troubleshooting Guide

Immunohistochemistry Troubleshooting Guide

Tips for troubleshooting immuohistochemistry assays. Top tips include solutions for no staining, high background and non-specific staining.

Immunostaining Schematics

No Staining

Reason Explaination
The primary and secondary antibody don’t match.
The secondary antibody must be against the species the primary antibody was raised in – anti that species
Is the antibody suitable for IHC protocols.
The antibody used may not recognise the native protein form. Test on a non-denatured and denatured western blot to ensure the antibody is recognizing non-denatured antigen
The secondary antibody wasn’t stored in the dark.
It is recommended to avoid exposing the secondary antibody to light. Store in the dark
Not enough primary antibody is binding the protein of interest.
Used a more concentrated antibody solution or increase incubation time
The protein of interest is not abundantly present in the tissue.
Use a more concentrated antibody solution
The protein of interest is a nuclear protein and the antibody cannot penetrate the nucleus.
Add a permeabilization step so the antibody can bind the antigen
Insufficient deparaffinization.
Incubate for longer in xylene. Always use make up fresh xylene solutions.
Formalin may be masking the antigen.
Use antigen retrieval methods
PBS contamination.
Use a preservative such as 0.01% azide in the PBS antibody storage buffer alternatively use fresh sterile PBS

High Background

Reason Explanation
Insufficient blocking.
Increase the blocking incubation period
Primary antibody used is to concentrate.
Use a more dilute concentration
Nonspecific binding of secondary antibody.
Run a secondary control without primary antibody
Inadequate washing.
Wash with PBS between all steps
Endogenous peroxidase activity.

Use inhibitors such as H2O2

Too much substrate added.
Reduce substrate incubation time
Chromogen reacts with PBS.
Use a Tris buffer to wash instead of PBS
Permeabilization has damaged the membrane and removed the membrane protein.
Remove permeabilizing agent from your buffers

Non-specific Staining

Reason Explanation
The concentration of the primary or secondary antibody is too high.
Try decreasing the antibody concentration and/or the incubation period.
The primary antibody is raised against the same species as the tissue stained (e.g rabbit primary antibody tested on rabbit tissue).
Use primary/secondary antibodies raised against a different species than your tissue
Endogenous peroxidase activity.

Use inhibitors such as H2O2

The sections/cells have dried out.
Do not let tissue sections dry out