High-pressure freezing, chemical fixation and freeze-substitution for immuno-electron microscopy

This chapter deals with tissue preparation for subsequent detection of molecules in biological samples using immunocytochemistry and transmission electron microscopy. The aim of these methods is to localize specific molecules at high resolution in order to identify their subcellular (or exact extracellular) localization. The methods are based on the use of antibodies or other affinity markers that bind specifically to a molecule of interest and a suitable detection system, e.g. a secondary antibody coupled to a gold particle of 5-15 nm size. Two different ways of sample preparation are described: (1) high-pressure freezing followed by freeze-substitution and immunogold labeling and (2) chemical fixation followed by freeze-substitution and immunogold labeling. Both methods have advantages and disadvantages that influence their utility in a given study design.