Immunocytochemistry tissue sections

Nakai Y, Shioda S, Ochiai H, Kudo J, Hashimoto A (1983) Ultrastructural relationship between monoamine-and TRH-containing axons in the rat median eminence as revealed by combined autoradiography and immunocytochemistry in the same tissue section. Cell Tiss. Res 230 1-14. 

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. 

Here then is how it all works together. Immunocytochemistry takes tissue sections or culture cells and incubates them with antibodies. The experimental needs determine the exact order of antibodies incubations and the specific labeling of the antibodies. The general steps in a single primary (1°) antibody indirect immunocytochemistry experiment include the following ... 

Human tissue should be fixed in 10% neutral buffered formalin and then dehydrated for embedding in paraffin. Paraffin is nonaqueous embedding medium, so the tissue blocks must have the water removed or be dehydrated. Dehydration is done in organic solvents such as alcohol, acetone, xylene, or toluene. After dehydration, the tissue blocks are embedded with liquid (warm) paraffin. When cooled, the wax embedded block is sectioned on a rotary microtome. Before immunocytochemistry can be performed on the resulting tissue sections, they must be rehydrated by processing with the same organic solvents back to water. Thus, the dehydration and rehydration steps are needed before immunohistochemistry. 

For antibodies to penetrate inside fixed cells, the membranes must be opened with detergents. Membranes are lipid bilayers that have a hydrophilic or water-soluble side facing the cytoplasm and the extracellular space (Fig. 5.4a). The hydrophobic or water-insoluble sides face each other at the center of the membrane. Also, there are transmembrane proteins that interact with the lipids and are held in the membrane. Membranes are barriers because they do not allow water or hydrated molecules to pass. For immunocytochemistry, the membrane must be parhally dissolved to allow antibodies to cross. It is also important that the transmembrane proteins remain cross-linked to other proteins so that they are not washed away (Fig. 5.4b). Detergent will dissolve the membranes but not the transmembrane proteins, which are cross-linked by the fixative to other proteins (e.g., scaffold proteins). For tissue sections, antibodies must penetrate through many cell layers into the center of the section. Achieving this depth of penetration requires removing most of the cell membranes but leaving the proteins so that they can bind antibodies when needed. 

In contrast, when using fluorescence immunocytochemistry to label proteins in tissue sections, the absolute relationship of fluorescence intensity to protein abundance can only be determined through conducting rigorous control experiments, and thus linearity should not be presumed. With that said, because CCD and photomultipHer tube detection systems are linear across a broad range of fluorescence intensities [36], quantitative fluorescence microscopy has been used in numerous smdies to measure relative changes in protein level [33, 35, 36]. In a system where the expression of a protein is only effected in a subpopulation of cells (e.g., GAD67 expression is reduced in a subset of PV+ interneurons in schizophrenia—see ref. 45), quantitative fluorescence immunohistochemistry will be more informative than techniques (e.g.. Western blot) that treat the tissue as a whole. 

Shiurba RA, Spooner ET, Ishiguro K, et al. Immunocytochemistry of formalin-fixed human brain tissues microwave irradiation of free-floating sections. Brain Res. Brain Res. Protoc. 1998 2 109-119. 

Immunocytochemical methods have been widely applied to visualize proteins, carbohydrates, or lipids in sectioned material. The advantage of using immunocytochemistry is to be able to localize the molecules of interest within the tissue. Several procedures have been described. Basically, these procedures can be split into four main steps that are described in subheadings (1) tissue preparation, (2) the primary antibodies, (3) the visualization of the target, and (4) enhancement of signals with antibody complexes. In addition, a protocol for alkaline phosphatase will be presented in detail in Subheading 5. The terms primary and secondary antibodies refer to the order in which they are applied to the target. The immunocytochemical procedures are not limited to sectioned... 

Sectioning material for immunocytochemistry with glass and diamond knives is identical to the process used for conventional EM. One difficulty frequently encountered is that unosmicated tissue embedded in plastic is sufficiently cleared by the solvent and embedding process as to be almost invisible. This makes orientation of the tissue in the trimming of the blocks difficult and increases the chances of mistakes of cutting too much. A small amount of Sudan Black dye can be added to... 

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... 

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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