High background in immunohistochemistry

Explore causes of high background in immunohistochemistry

Why is there high background in my IHC experiment?

Possible causes

Solution

Blocking of non-specific binding might be insufficient

Increase the blocking incubation period and consider changing the blocking agent. We recommend 10% normal serum of the species of the secondary antibody for 1 hr.

If using an amplification technique, signal amplification may be too high

Reduce the amount of signal amplification (eg conjugate less biotin to secondary antibody if using biotinylation).

If using enzyme-conjugated antibody, endogenous enzymes are active

Endogenous enzymes may give false positives, so you will need to block them with inhibitors prior to immunostaining.

For alkaline phosphatase (AP conjugated antibodies), use Levamisol (2 mM).
For biotin-based IHC detection, use our Avidin/Biotin Blocking Kit.
For peroxidase (HRP conjugated antibodies), use or H₂O₂ (0.3% v/v), or our ready-to-use Hydrogen Peroxide blocking agent.

If using formalin / PFA fixatives and fluorescent detection, fixative is giving a fluorescent background signal

Formalin/PFA usually fluoresce at green wavelengths, so try a fluorophore in the red or infared range to minimize overlap.

Incubation temperature may be too high

Make sure you use a secondary antibody raised in a different species to your sample.

Primary antibody concentration may be too high

Dilute the antibody further to its optimal concentration

The secondary antibody may be binding non-specifically

Run a control without any primary antibody.
Make sure you use a secondary antibody raised in a different species to your sample.
Try a secondary antibody that has been pre-adsorbed against the lg of the species of your samples.

The tissue sections have dried out

Examine the tissue; sections with higher background staining at the edges than towards the centre are often dried out.

Prevent tissue sections drying out by keeping them in a humidified chamber.

Tissue not washed enough

Residual fixative or unbound antibodies remaining between steps can produce a false positive signal.

Increase the washing time
Wash the tissue extensively in buffer between all steps.

If using enzyme-conjugated antibody, too much substrate

Dilute the substrate and reduce substrate incubation time.

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