Fixation and Embedding

For electron microscopic immunocytochemistry, the fixation procedure is always a compromise between good morphological preservation and retention of antigenicity (1 , 2 ). It is preferable to fix tissues by perfusion, but if that is not possible, the time between removal of the tissue and fixation should be kept as short as possible. The fixative used will depend on whether the immunogold labeling will be done before or after the samples are embedded and on how resistant the antigen is to the fixative. In order to preserve the ultrastructural detail, it is desirable to include glutaraldehyde in the fixative. However, some antigens are extremely sensitive to crosslinking by glutaraldehyde, and the concentration of glutaraldehyde will have to be reduced or, in the most extreme cases, omitted entirely. For pre-embedding staining, it is possible to immunolabel the samples prior to fixation or after only a very mild fixation. Following incubation in the primary antibody or at subsequent steps, the samples can then be refixed in a stronger fixative, such as 2% glutaraldehyde, to give good morphological preservation. For postembedding labeling, it is not possible to refix the tissue after immunolabeling. Therefore, the composition of the initial fixative must be such that morphological detail and antigenicity are both preserved. The composition of the fixative will have to be determined empirically for each antigen.