Immunohistochemical Analysis of Transgene Expression
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 (1; see Note 1). 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 (2), 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 (3). 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 (see Note 2).
KeywordsNickel H2O2 Filtration Paraffin Hematoxylin
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