Introduction to Immunocytochemistry

 

Introduction to Immunocytochemistry

I mmunocytochemistry is the name given to methods that use antibodies to detect the location of proteins within cells. The antibodies bind specifically to the protein being investigated. With electron microscopes this method is used with electron opaque markers which bind to the antibodies and visualize their location within the cell. One useful electron opaque marker is colloidal gold .

For electron microscopy it is important to have preparation methods that open the cells and allow access of antibodies to the proteins (or antigens) they bind to without destroying the normal cellular organization (or morphology).

Over the last ten years, specimen preparation techniques, especially those for immunocytochemistry, have reached high levels of sophistication and allow cells to be opened, labeled with antibodies and colloidal gold and yet retain good morphology. Some of these methods are briefly described in our introduction to electron microscopy.

At the moment, the very best way to gain access to the inside of cells is by cutting sections through them. In this way the morphology is retained but antibodies gain total accessibility to the cut face of the cell. To cut sections thin enough to be examined in the electron microscope (approximately 40 - 100 nm), the cells must be made hard, either by embedding in Aresin or by freezing them in a cryoprotectant. Both methods are used routinely in the Center for Cell Imaging at Yale. For antigens which are only present in small amounts in the cell the most sensitive method is usually to label sections of frozen material.