Immunohistochemistry (IHC) is a critical technique in both clinical and life science research laboratories, combining the power of specific antibodies and microscopy to reveal both the location and abundance of a given protein in a tissue section.
In theory, the protocol is straightforward: A thinly sliced tissue sample, either flash-frozen or formalin-fixed and paraffin-embedded, is placed on a microscope slide and incubated with an antibody specific to the protein of interest followed by signal detection using a chromogenic substrate or fluorescent dye. The signal is observed microscopically in areas where the antibody is bound to the protein of interest.
In reality, optimizing the protocol steps for an IHC assay involves managing several key parameters and can be labor intensive. While a standard protocol may work very well with some antibodies, the same protocol may not work perfectly with all antibodies, and certain antibodies may require protocol modifications in order to generate optimal results.
Here, we outline some of the key considerations users should bear in mind when setting up their own IHC experiments.
1. Understand your antibody
Commercial antibodies often come with datasheets that assert the antibody works for IHC and include a color image of a stained tissue section, however, it is still essential to perform experiments to confirm antibody specificity in your cell type. It is equally important to characterize homemade antibodies rigorously to ensure antibody performance and specificity.
We recommend users test their commercially purchased or homemade antibodies by western blotting before applying them to precious tissue sections. Relevant positive and negative cell lysates should be used to ensure that the antibody performs specifically and appropriately. Having verified antibody specificity by western blot, the user can test the antibody for IHC by titrating on known antigen-positive and antigen-negative cell models, e.g. paraffin embedded cell pellets. (You can prepare these paraffin-embedded cell pellets as you would a tissue section by scraping cells off a culture plate, fixing, processing and embedding them in paraffin.) Among other things, this step will help you to determine a working antibody concentration/dilution.
Antibody specificity should also be assessed by testing on a tissue microarray comprised of relevant tissue cores. These arrays are available commercially from various suppliers or can be built from in-house tissue banks. Finally, if the antibody was raised against a peptide antigen, a peptide blocking experiment will demonstrate antigen specificity.
2. Phospho-specific considerations
If your antibody is directed against a phosphorylation site, you will need to confirm modification-specificity of the antibody as part of the antibody validation process. To confirm phospho-specificity, treat a serial tissue section with phosphatase to remove the phosphate groups. Test the antibody on the tissue in the presence or absence of phosphatase treatment to ensure no staining is detected in the phosphatase-treated section.
If your antibody specifically targets a phosphorylated receptor tyrosine kinase, such as Her2, a western blot assessing a panel of phosphorylated tyrosine kinases will help identify cross-reactivity. Though it’s impossible to assess every possible cross-reactive protein – there are simply too many antigens to test – this preliminary step is a good gauge of antibody promiscuity.
Phospho-specific antibodies require a little extra attention during IHC protocol optimization, too. Sodium citrate is generally a good buffer for antigen retrieval, but antibodies directed against phospho-tyrosine often perform more successfully with EDTA retrieval. Likewise, blocking solutions with casein generally work well, but use of these reagents in conjunction with phospho-specific antibodies may result in diminished signal. Tris-buffered saline with Tween®-20 (TBST) and goat serum blocking solution does not interfere with phospho-specific signal, making it a good alternative to traditional casein blocking.
Unfortunately, there is no easy path to optimizing an IHC experiment. The variables can seem endless, from the buffers used in antigen retrieval, to the choice of blocking solutions, to the antibody concentration.
Many companies provide protocols identifying the key parameters and reagents used during in-house validation of the antibody for IHC, such as the antibody diluent. Researchers often use a common diluent such as TBST, but the choice of diluent can have a significant impact on the performance of an antibody. Minimize troubleshooting by following the manufacturer’s diluent recommendations, when available.
Similarly, the choice of detection system can significantly impact staining results as well. Traditional detection systems typically employ avidin-biotin. Newer detection systems use an enzyme-conjugated polymer reagent, which generally produces a cleaner, stronger signal and requires less antibody. For example, if your vendor-supplied protocol recommends a 1:500 antibody dilution using the polymer-based detection system, the same results with avidin-biotin may be achieved, but further optimization of the antibody concentration will likely be required.
Homemade antibodies, of course, require extensive protocol optimization and several protocols are available online. Cell Signaling Technology offers two protocols on its website, as well as a troubleshooting guide [http://www.cellsignal.com/support/protocols/IHC-troubleshooting-guide.html] to enable researchers to perform successful IHC experiments.
It might take several weeks to establish antibody specificity and to optimize the staining protocol, but the time invested in ensuring specific and reproducible results may save you months of aggravation down the line.
Katherine Crosby, Manager, Immunohistochemistry Group, Cell Signaling Technology
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