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 an enzyme-conjugated antibody, endogenous enzymes may be active
These endogenous enzymes can give false‑positive results, so they must be blocked with appropriate inhibitors prior to immunostaining.
- To block endogenous alkaline phosphatase ( for AP-conjugated antibodies), use Levamisol (2 mM) (available in our catalogue as ab141217, Levamisole hydrochloride, Alkaline phosphatase inhibitor).
- To block endogenous peroxidase (for HRP conjugated antibodies), use H2O2 (0.3% v/v), or our ready-to-use Hydrogen Peroxide blocking agent.
Variations:
- For fragile samples, where preservation of the morphology is required, dilute the hydrogen peroxide in methanol.
- For cell surface or membrane proteins, use hydrogen peroxide in PBS or quench with peroxide after first incubating sections or cells with the primary antibody.
If using an enzyme-conjugated antibody, too much substrate
- Dilute the substrate.
- Reduce substrate incubation time.
If using a biotin–based detection system, endogenous biotin within the tissue may be binding to streptavidin or avidin
- Use our Avidin/Biotin Blocking Kit as an additional blocking step.
If testing a mouse primary antibody on mouse tissue, the secondary antibody may be binding to endogenous mouse immunoglobulins and Fc receptors
- Follow a Mouse-on-mouse (MOM) staining protocol.
If using formaldehyde-based fixatives and fluorescent detection, the fixative may give a fluorescent background signal
- Formalin/PFA usually fluoresce at green wavelengths, so try a fluorophore in the red or infrared range to minimize overlap.
Incubation temperature may be too high
- If incubating the primary at RT, try incubating instead overnight at +4 °C.
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 from your sample.
- Try using a secondary antibody that has been pre-adsorbed against the immunoglobulin of the sample species.
- Dilute the antibody further to its optimal concentration.
The tissue sections may 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 from drying out by keeping them in a humidified chamber.
- Use a hydrophobic barrier pen to retain reagents in place, for example, ab2601, PAP pen.
- Keep tissue sections covered with buffer at all times.
The tissue may not be washed enough
Residual fixative or unbound antibodies remaining between steps can produce a false positive signal.
- Increase the washing time.
- Increase the number of washes.
- Wash the tissue extensively in buffer between all steps.