Immunohistochemistry troubleshooting: Five steps to publication-quality images
Tips for solving high background
Immunohistochemistry application, or IHC, offers a spatial visualization and location of an antigen in tissue sections. This application can be challenging, with problems generally falling into two categories: weak or no stain, and high background.
Here are some troubleshooting tips falling into the “high background”, for IHC:
- Insufficient washing can lead to high background due to traces of fixative.
- Be sure to wash thoroughly and repeat as directed.
- Inadequate fixation can lead to diffusion of antigen in the tissue.
- Be sure to follow fixation directions precisely.
- Diffuse staining can be caused by tissue damage; handle tissue carefully during fixation to preserve integrity.
- Insufficient penetration of detection reagents can be resolved by preparing thinner tissue sections.
- Different fixatives affect tissue antigen differently, so be sure to test and optimize the pH conditions, incubation time and temperature.
- Issues with the antigen retrieval step typically do not result in high background. However, it is important to use the proper antigen unmasking method, preferably via microwave, as prescribed by the fixation method.
- Possible reasons for high background that occur during the blocking step include interference from endogenous peroxidases or phosphatases, which can be resolved by quenching the enzymes with 3% hydrogen peroxide in methanol or water.
- A kit, such as Thermo Scientific’s Peroxidase Suppressor can also be used. Alternatively, treat by use of enzyme inhibitors for alkaline phosphatases.
- Another reason for high background can be interference from endogenous biotin activity. Treat this by blocking with the avidin-biotin blocking reagent before incubating primary Abs.
- Possible reasons for high background at the detection step can include non-specific binding of primary antibody - if the concentration is too high it elicits high-background and non-specific binding. Try using a higher dilution of the primary antibody.
- There can also be non-specific binding of secondary antibody, so try pre-treating with normal serum from the same species as the polyclonal antibody. You can also try increasing the incubation period.
- Secondary antibody can cause non-specific signal by reacting with tissue proteins. In this case it is best to switch to a pre-adsorbed, affinity purified secondary antibody, such as those offered by Thermo Fisher Scientific.
- If using antibodies of the same species, for example, mouse antibodies on mouse tissues; pre-treat the tissue with mouse-on-mouse blocking reagent.
- Sometimes high incubation temperatures can interfere with target detection. Solve this by lowering the temperature or even using a four-degree incubation.
- Some formalin or paraformaldehyde fixatives cause autofluorescence in the green spectrum influorescence IHC. Solve this by using a fluorophore in the red spectrum. If there is non-specific binding of polyclonal antibody, try using a monoclonal primary antibody instead of a polyclonal.
- High antibody concentration can cause non-specific binding. Decreasing the concentration of the primary and/or secondary antibody can solve this. Lastly, there could be a substrate problem. Reducing the substrate incubation, diluting the substrate, or changing to a different substrate source can address this.
- Once you perfected your staining, proceed to visualize your stained sample using the EVOS FL2 Auto Imaging System.
Immunohistochemistry can be a challenging application. These troubleshooting tips can help you achieve the publication-quality images you deserve.
Find recommended products and protocols at thermofisher.com/ihc5steps.