Basic Immunocytochemistry for Light Microscopy

Abstract

Immunocytochemistry, the identification of cell- or tissue-bound antigens in situ, by means of a specific antibody–antigen reaction, tagged microscopically by a visible label, has a remarkably wide range of applications. The basic techniques are straightforward and can be adapted to explore the localisation of virtually any molecule of interest to the researcher in samples of normal and/or malignant cells. Heterogeneity can be mapped and loss or gain of immunoreactivity with tumour progression can be visualised. In this chapter, methodologies are given for appropriate preparation of cells and tissues, including cells cultured on coverslips (which can be used for live cell imaging), cell smears, frozen (cryostat) and fixed, paraffin wax-embedded tissue sections. Heat- and enzyme-based antigen retrieval methods are covered. Basic detection methods, which can be readily adapted, are given for direct (labelled primary antibody), simple indirect (labelled secondary antibody), avidin–biotin (biotinylated primary antibody), avidin–biotin complex (ABC), peroxidase–anti-peroxidase or alkaline phosphatase–anti-alkaline phosphatase (PAP or APAAP), and polymer-based methods. The use of enzyme labels including horseradish peroxidase and alkaline phosphatase, and fluorescent labels, are considered.