Immunocytochemistry tissue dehydration

Opnns, A D., Geuze, H J, and Slot, J W (1994) Cryosubstitution dehydration of aldehyde-fixed tissue, a favourable approach to quantitative immunocytochemistry J Histochem Cytochem 42,497—503... 

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. 

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. 

The significant advantages of the described protocol rest in the versatility of probe design, the increased tissue permeability and hybridization efficiency because of the small probe size and, finally, the superior tissue preservation as a result of the rapid freezmg/dehydration and vapor-induced fixation. In addition, tissue preparation with the proposed protocol is compatible with immu-nocytochemistry. It is possible, therefore, to obtain serial consecutive paraffin sections from the same CNS and to combine in situ hybridization with immunocytochemistry for direct comparison of the signal from the two techniques in the same cells. For a more comprehensive and detailed review of in situ hybridization m general, the reader is referred to the excellent recent practical guide by Leitch et eil.(l) and also to the seminar series by Harris and Wilkinson (2). 

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