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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 (2mM).
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.