Immunocytochemistry standardization

In addition to the standard set of tissues specified in Table 7.8, observations during the course of the study or in other previous studies may dictate that additional tissues be collected or special examinations (e.g., special stains, polarized light or electron microscopy, immunocytochemistry, or quantitative morphometry) be undertaken to evaluate the relevance of, or understand the mechanisms underlying, certain observations. 

This method, either standard or elite (increased molar ratio), remains the forefront of much of the immunocytochemistry being performed today, and will be the main subject of this chapter. There are new methods, though, that are being used with increased frequency, such as the labeled-avidin binding method, sometimes called the streptavidin-binding method, and a newer catalyzed amplification method that uses avidin, biotin, peroxidase, and a biotinyl tyramide to achieve even more sensitivity. These methods will be discussed at the end of this chapter. 

Today, for research with animal tissue and cell cultures, the standard has become fixation in paraformaldehyde, with animal tissue sectioned in a cryostat, and then incubation of sections and cultures with antibodies. This book focuses on introducing the methods of immunocytochemistry for biomedical scientists. These chapters may be read in order for a complete understanding of immunocytochemistry, or the chapters may be read individually for information about specific topics. The book is designed to help the novice perform experiments, solve problems, get results, and understand more advanced texts when more advice is needed. 

The dehydration, embedding, and sectioning of pre-embedding electron microscopic immunocytochemistry blocks follow the standard electron microscopic procedures. However, for section staining, the time is reduced to 2-3 min to preserve the size of the silver particles. 

Currently, it is standard procedure to develop ion channel-specific antibodies for immunocytochemistry, to perform Western and Northern blot analyses, ion channel in situ hybridization, or reverse transcription polymerase chain reaction (RT-PCR). The introduction of the single-cell RT-PCR in combination with the patch-clamp method in the 1990s made it possible to identify gene transcripts and to correlate them with functional data for the same individual cell. Finally, one of the most powerful cell biological techniques in the study of ion channels is based on artificial expression systems such as microinjection of mRNA encoding channel subunits into Xenopus oocytes and selective expression of native ion channels or with different subunit composition (e.g., Ky channel subunits). Because the Xenopus oocytes are large, they are a perfect model to study artificially expressed channels. Another good model for artificial ion channel expression is the Chinese hamster ovary (CHO) cell line. 

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