Pre- or postembedding immunocytochemistry: which to choose for the localization of glial fibrillary acidic protein (GFAP)?

Immunostaining for glial fibrillary acidic protein was performed in the hippocampus and cerebellum of adult rats in order to compare the distributions of immunolabelling after pre- and postembedding procedures. The reactions of protoplasmic astrocytes and pericapillary astrocyte processes were investigated at the electron microscopic level. After the pre-embedding reaction, visualized with 3,3'-diaminobenzidine tetrahydrochloride, a granular precipitate was observed to decorate the rough endoplasmic reticulum of the astrocyte cell bodies and a precipitate filled the cytoplasm in the astrocyte processes. In studies with the postembedding procedure, immunogold particles were observed to be attached exclusively to the intermediate filaments of the astrocytic cytoskeleton both in the cell body and in the processes. It is concluded that the diaminobenzidine precipitate diffuses in the cytosol, pre-embedding immunocytochemistry is therefore, suitable for the overall labelling of astrocytes, whereas the postembedding procedure reveals the exact intracytoplasmic localization of glial fibrillary acidic protein. This explains the similar pre-embedding immunostaining patterns of astrocytes with markedly different amounts of glial filaments (e.g. protoplasmic, fibrous and reactive) and stresses the importance of the use of the postembedding method when an exact intracellular localization is required. The results also suggest that, in spite of claims of soluble cytoplasmic pools of this protein, the glial filaments are the exclusive domains of immunoreactive glial fibrillary acidic protein.