engleski

Effects of Microwave Heating on Antigen Retrieval in Cryosections of Formalin-Fixed Rat Tissues

Immunocytochemistry is a method for localizing proteins in the mammalian tissues and cultured cells. Samples for immunocytochemical studies are often fixed with formalin (2%-10% p-formaldehyde), frozen or embedded in paraffin, and sectioned. Fixation with formalin denatures proteins while preserving antigenicity, but it can ‘’mask’’ the antibody binding sites (epitopes), thus diminishing the availability of epitopes and fidelity of immunocytochemical findings. Hidden epitopes can be recovered by various antigen retrieval protocols before applying antibodies. These protocols are normally used with paraffin sections, and include heating in defined buffers, treatment with various alcohols, detergents, metals or high pressure, or various combinations of these procedures. Here we report on using similar antigen retrieval protocols on cryosections of the formalin-fixed rat kidney and liver tissues in order to reveal optimal immunostaining conditions for representative proteins localized in the cell membrane (Na/K-ATPase, aquaporin 1, megalin, cell adhesion molecule CAM105), cytoplasm (metallothionein), intracellular organelles (vacuolar H+-ATPase), and cytoskeleton (actin, tubulin). Our immunocytochemical data illustrate that: a) harsh antigen retrieval protocols, previously used with sections of the formalin-fixed paraffin-embedded tissues, can be efficiently used with cryosections of the formalin-fixed tissues, b) for each set of antigen-antibody, it is always necessary to test various antigen revealing protocols in order to determine optimal conditions for immunocytochemical presentation, and c) in most cases the protocols that include microwave heating give the best unmasking effects and staining intensity. Being the strongest unmasking method for different epitopes, microwave heating also generates conditions for significant reduction of the amount of primary antibodies.

antigen retrieval; cryosections; immunocytochemistry; microwave heating; formaline fixation; rat; kidney; liver

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