Preembedding immuno-staining for electron microscopy
Wolf D. Kuhlmann
Division of Radiooncology, Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany
The localization of intracellular antigens by labeled antibodies is governed by their penetrability. Studies with aldehyde fixed cell suspensions and various marker molecules of known molecular weight have shown that molecules of 80,000 daltons and more do not penetrate cell membranes or do so only sluggishly. Enzymatic predigestion of cell surface material improved penetration, however, the total number of stained cells remained low. In order to overcome penetration problems, treatments of aldehyde fixed cells f.e. with saponin, Tween and other detergents were examined. Yet, no convincing results could be obtained with hand-cut tissue fragments.The best results were obtained with thick frozen sections from well-fixed specimens. The adoption of cryomicrotomy to preembedment immunohistology is not difficult, and a typical protocol is described for the immunoperoxidase detection of alpha-1-fetoprotein in rat liver (fetal liver; regenerating liver after injury; hepatoma bearing liver).