Immunohistochemical analysis of transgene expression.

Abstract

Immunohistochemistry can be used to localize the peptide or protein product of the transgene, or to identify particular cell types within tissue sections by labeling a specific protein. There are many ways to do this. Today many involve a primary antibody to the protein of interest, and a secondary antibody plus a fluorescent or enzyme tag to visualize the reaction. The fluorescent tag is observed by viewing it through a microscope equipped with a light source of the correct wavelength to excite the molecules of the tag, which then emit light of a specific wavelength, visible through filters. Because the fluorescent material contains a limited number of molecules, the label is temporary and needs to be photographed to save the result obtained. The second protocol given here uses a fluorescent tag. In contrast, the enzymes alkaline phosphatase and horseradish peroxidase can be visualized with chromogens that create a permanent slide, which can be examined immediately in a simple bright-field microscope and reviewed later. Additionally, it allows a clear bright-field or Nomarski view of the tissue, together with the label. These approaches give a one-enzyme molecule for one antibody-antigen reaction. For proteins in low abundance, the reaction may be enhanced by Sternberger's peroxidase antiperoxidase (PAP) method, which attaches a three-enzyme complex to the secondary antibody. Alternatively, the enhancement is accomplished by using a biotin-labeled secondary antibody that binds a streptavidin molecule complexed to enzyme molecules. The latter approach is the first method described here. It has yielded good results in a variety of tissues, and, with the correct dilution of the antibodies and streptavidin complex, results in a clear signal.