Fixation and embedding.

For electron microscopic immunocytochemistry, the fixation procedure is always a compromise between good morphological preservation and retention of antigenicity. The choice of fixative depends on whether the immunogold labeling will be done before or after the samples are embedded and on how resistant the antigen is to fixation. For preembedding staining, it is possible to immunolabel the samples prior to fixation or after only a very mild fixation. Following immunolabeling, the samples can be refixed in a stronger fixative, such as 2% glutaraldehyde, to give good morphological preservation. Since it is not possible to refix the tissue after immunolabeling, for postembedding labeling, the composition of the initial fixative must be such that morphological detail and antigenicity are both preserved. While virtually any embedding resin may be used for immunogold staining, for postembedding methods, the resin can affect the immunostaining. In this chapter, methods are given for conventional fixation and microwave fixation as well as for embedding in various resins.