Immunocytochemistry animal tissue research, methods

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. 

For immunocytochemistry, fixed tissue must be cut in thin sections to be viewed in the light or fluorescence microscope. There are two common ways of sectioning tissue - the cryostat for fixed frozen tissue and the rotary microtome for paraffin-embedded tissue. In animal research, select the sectioning method based on the experimental design. The method that gives the most reliable results and is the simplest should be selected. For immunocytochemistry, the cryostat is a very efficient and reliable method. The rotary microtome of paraffin-embedded material is more complex and problematic. For immunocytochemistry in animal research, the cryostat method is recommended for reasons discussed in this chapter. 

Traditionally, formalin-fixed and paraffin-embedded tissue is used by pathologists for examining human tissue. This method was first adapted for immunocytochemistry with tissue from research animals. Today, for animal research, there is no requirement to use formalin-fixed and paraffin-embedded tissue methods. Table 4.1 shows the steps necessary for formalin/paraffin immunohistochemistry... 

This new definition of immunocytochemistry derives from advances in antibodylabeling methods in recent years. These advances resulted from specific needs in animal research. Initially, formalin-fixed paraffin sections were used for immuno-histochemistry however, results were inconsistent. In most cases, the antibody did not label anything or it labeled too many cells and was dubbed over fixed. This problem led to the development of the epitope retrieval or antigen retrieval methods, where sections of tissue are treated with heat in buffers before antibody incubations. Unfortunately, epitope retrieval methods can be unique from antibody to antibody and also, for the same antibody, from tissue to tissue. Epitope retrieval is complicated and best avoided. For animal research, a simple method was then developed where tissue was fixed in paraformaldehyde and not formalin or alcohol and subsequently frozen sections were cut on a cryostat. This eliminated the steps of dehydration, embedding in paraffin, rehydration after sectioning, and epitope retrieval before antibody incubation. This was a major breakthrough. 

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