Immunocytochemistry
Immunocytochemistry is the use of antibodies for identifying proteins and molecules in cells and tissues viewed under a microscope. Immunocytochemistry harnesses the power of antibodies to give highly specific binding to unique sequences of amino acids in proteins. Perhaps the most exciting part of using antibodies is that new antibodies can be generated on an as-needed basis, thus providing a constant source of new reagents. Scientists are constantly generating new antibodies to specific parts of molecules thus driving continual evolution of immunocytochemistry. Identifying the location of antibodies in cells is based on availability of labels that is, itself, rapidly advancing. As time passes, immunocytochemistry continues to respond to new development of labels and advanced methods of labeling molecules.
If the terms immunocytochemistry and immunohistochemistry seem similar then here is why. Many years ago, immunocytochemistry was defined as the use of antibodies to study cells in the form of cultures or smears from animals. Immunohistochemistry, on the other hand, was defined as the use of antibodies to study paraffin sections from human tissue. Today, immunohistochemistry is still the use of antibodies in paraffin sections in human pathology, but the definition of immunocytochemistry has changed. Immunocytochemistry is the use of antibodies in animal research with cells and tissues fixed in paraformaldehyde.
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 immunohistochemistry; 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.
What Can Immunocytochemistry Tell Us?
Immunocytochemistry harnesses antibodies that are specific reagents and which allow unique detection of proteins and molecules. Using antibodies requires specific methods, labels, and controls. Performing immunocytochemistry experiments requires some basic knowledge of biology.
Much of the data collected in biomedical research today results from biochemical and molecular methods, where many cells are pooled for analysis. For example, enzyme assay of the liver will give values that, when repeated, should be statistical similar and should provide reliable average values with standard errors. When this and similar methods pool many cells for analysis, they are broadly defined as “population studies”. However, problems result, because not all liver cells might have the specific enzyme of interest. So changes found with the enzyme assay might be due to enzyme activity in all of the liver cells or might be due to enzyme activity in only some of the cells. Rather than assuming all of the cells in the liver have the enzyme, the complementary approach is to look at the cells with morphological methods.
Morphological approaches in biomedical research can include a wide range of microscopes, but today typically employ immunocytochemistry that can give us information about individual liver cells containing the specific enzyme. Immunocytochemistry uses antibodies to bind proteins and labels to show protein’s location. If, for example, the enzyme is a marker for inflammation, then the location of cells with this enzyme tells us which cell types have the inflammatory response. Thus, immunocytochemistry methods are broadly defined as “individual studies” of single cells or cell groups. The resulting data tell us about location of the enzyme.
Reference:
Burry, R. W. Immunocytochemistry. A Practical Guide for Biomedical Research. 2010.