Immunohistochemical detection of Bcl2 family proteins in tissues

The approach we prefer for immunohistochemical detection of Bcl-2 protein is to employ sections derived from paraffin-embedded tissues (Figure 1). This yields morphology which is far superior to cryostat sections and permits use of

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Figure"!. Irnmunohistochemical analysis of Bcl-2 family protein in tissue sections.

Examples are provided of irnmunohistochemical analysis of Scl-2 (A-C). Mcl-1 (D-F), and

Bcl-X (G-l) in lymph nodes. Photomicrographs are presented at low (first column).

medium (second column), and high (third column) magnification. Immunodetection was accomplished by a diammobenzidine method (brown) and nuclei were counterstained with haematoxylin (blue). The tissue sections highlight the different patterns of Bcl-2

family protein expression in the germinal centre (GC) lymphocytes of the lymphoid follicles and the surrounding mantle zone region and interfollicular zones. (A-C) Bcl-2

staining is found primarily in the mantle zone lymphocytes surrounding the germinal centres and in occassional cells in the interfollicular regions. (D-F) IVlcl-1 immnostaining germinal centre lymphocytes. (G-l) Bcl-X staining is found primarily in is strongest in plasma cells and occassional activated lymphocytes.

archival clinical material. A critical aspect of the procedure is either fixation of the tissues in an acidic fixative or, if fixed in a neutral solution, subsequent treatment with an acidic solution. The lower pH somehow reveals the epitopes that are recognized by all of the currently available commercial antibodies, thus markedly improving sensitivity. This same approach has also yielded superior results for antibodies raised against other members of the Bcl-2 protein family, such as Bcl-X, Mcl-1, and Bax (12-19). Several colori-metric or fluorescence-based detection systems can theoretically be employed for visualization of the antibodies, but most of our experience is with horseradish peroxidase (HRPase)-based colorimetric detection methods.

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