미세구조적 면역세포화학법을 위한 포매전 염색법

Abstract

Immunocytochemistry is an important technique for studying location and function of specific proteins and certain other macromolecules such as nucleic acids and polysaccharides in cells and tissues. Immunocytochemistry is based on the coupling of antibodies to specific antigen. Immunocytochemical methods can be divided into two classes, preembedding methods and postembedding methods. Preembedding methods are those in which the cell, or isolated organelle, is labeled before embedding and sectioning. For preembedding method, periodate-lysine- paraformaldehyde, 4% paraformaldehyde, or 4% paraformaldehyde plus 0.1% glutaraldehyde. While the outside layer of the cell may be accessible to antibodies, the labeling of intracellular structures must be preceded by a solvent or detergent(e. g. triton X and saponin) permeabilization step. Optimal staining for primary antibody is generally obtained by overnight incubations at 4°C. For increasing secondary antibody penetration, we use the peroxidase conjugated Fab instead of than the complete IgG. The 3, 3'-diaminobenzidine(DAB) has been widely used as electron donor for ultrastructural. After postfixation with 1% glutaraldehyde and 2% osmic acid, tissue is embedded in Epon 812 using a two-stage flat embedding procedure which allows easier handling of material and correlative microscopic analysis.