Processing of most samples starts with fixation to preserve morphology.

A fixation method must be chosen that balances two characteristics: good preservation of cellular 3-D structure and adequate access to antigenic sites.

The goal is to preserve sufficient cellular organization to allow identification of the features of interest, but not to destroy the antigenicity of the target. Fixation is also frequently combined with permeabilization to allow the staining solutions used in later steps access to the cytoplasm.

Commonly used histological methods of fixation and permeabilization often consist of treating the cells with solvents, such as alcohol or acetone. While these methods are quick acting precipitating fixatives, they are also good permeabilizing agents, but have one significant negative consequence: cellular shrinkage. The degree of shrinkage may be almost insignificant for monolayers of cells, but will distort tissue samples dramatically. To provide the ability to take full advantage of the three-dimensional reconstruction capability of the confocal microscope, the use of a fixative that does not destroy in vivo structure and organization must be found.

Glutaraldehyde (GA) is a commonly used fixative for electron microscopy, and does a terrific job to preserve microscopic structures. Unfortunately, glutaraldehyde has the side effect of cross-linking many epitopes; this may render the tissue undetectable by antibodies. An additional undesirable side effect is the enhanced autofluorescence from residual unbound free aldehydes.

A good compromise is the use of paraformaldehyde (PFA) as a fixative. PFA will preserve most structures detectable at the confocal microscope level. Also, it will usually not obscure the epitopes of interest.

It is important to remember that different specimens may require different fixation methods. Testing and optimizing for each new sample type will insure that the best balance between preservation and labeling is obtained.