Immunocytochemical demonstrations of cytoplasmic and cell‐surface EGF receptors in A431 cells using cryo‐ultramicrotomy, surface replication, freeze‐etching and label fracture

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Abstract
SUMMARY: In this paper we describe the use of a number of complimentary methods to visualize cytoplasmic and cell‐surface located epidermal growth factor (EGF) receptors in cultured A431 cells. Cryo‐ultramicrotomy in combination with immuno‐gold labelling will be shown to provide an excellent method in visualizing cytoplasmic located EGF receptors in addition to cell‐surface located EGF receptors. An important aspect in this method involves the possible effects of the fixatives on antigenicity. Using radioactive labelled anti EGF receptor antibodies, it was shown that formaldehyde as a fixative had no significant effect on label‐efficiency.The density and lateral distribution of EGF receptors at the cell surface has been studied by three methods, i.e. surface replication, freeze etching and label fracture, all methods in conjunction with immuno‐gold labelling. These methods allow in principle a quantitation of the surface distribution of the EGF receptors. The surface‐replication method involves, however, dehydration and critical‐point drying steps, and using radioactive labelled anti EGF receptor antibodies it was shown that in particular OsO4 fixation and dehydration caused a significant loss of cell‐associated antibodies. This disadvantage is overcome by freeze etching and the label‐fracture method, and as such these techniques provide the best methods for quantitative analysis of the planar distribution of cell‐surface located EGF‐receptors.