IHC

By Jamshed Arslan, Pharm D, PhD

By Jamshed Arslan, Pharm. D., PhD.

Signal amplification methods are widely used in immunohistochemistry (IHC) for detection of rare epitopes and low abundance antigens. While many of these techniques such as the avidin-biotin complex (ABC) method improve staining, they frequently require additional steps and result in higher background staining.  Blocking endogenous biotin, a requirement of using ABC reagents, may not sufficiently remove residual activity in frozen tissue sections and tissues high in biotin including the liver and kidney.

There are a variety of experimental methods to choose from when using antibodies as a probe to highlight a target of interest. Western blot will reveal protein abundance and behavior, immunocytochemistry allows a look at protein behavior on the cellular level, and flow cytometry has the ability to label and sort hundreds of thousands of cells in no time. However, immunohistochemistry (IHC) is the only method where researchers have the ability to view the spatial localization of a target within a specific tissue.

Immunohistochemistry (IHC) is a widely applied experimental method used to examine tissue antigen expression and behavior with the use of an antibody conjugated to a secondary tag for visualization.  IHC consists of a tissue preparation phase, an antibody-staining phase, and a result analysis phase - all of which may lead to skewed results if not properly performed.  One way to ensure that IHC staining results are in fact demonstrating protein behavior and not a side effect the experimental process is to use IHC controls within each expe

Even though the direct detection method is becoming more popular for immunofluorescence (IF) and flow cytometry experiments, the indirect detection method still remains the preferred choice for many other applications. In direct detection, the labeled primary antibody is responsible for both binding and detection of the antigen of interest.

Interpretation of immunohistochemistry (IHC) data is difficult in the absence of appropriate controls.

To confirm staining specificity and instill confidence in your results, a positive and negative tissue control should be routinely included in IHC experiments.

Fluorescent probes conjugated to antibodies allow for simultaneous IHC detection of multiple antigens in the same tissue section. However, quite often conventional multi-color IHC cannot be done if only primary antibodies raised in the same host species are available to the researcher. To solve this problem, we developed a novel technique for performing multicolor fluorescence immunohistochemistry using primary antibodies derived from a single host source.