Methodological basis for an autoradiographic demonstration of insulin receptor sites on the surface of whole cells: a study using light‐ and scanning electron microscopy

Abstract

SUMMARY An autoradiographic technique is described which allows, on whole cells, the determination of the number of receptor sites which are occupied by 125 I‐insulin according to defined binding conditions. Glutaraldehyde fixation prevents the dissociation of the bound tracer from the receptor, which is the basis for a quantitative correlation between biochemical and autoradiographical data. The homogeneous coating of whole cells with a thin emulsion layer, which was required for these experiments, was achieved by a stripping film technique. An accurate judgment of the coating was possible only with scanning electron microscopic (SEM) techniques. As observed in the SEM, the photographic layer was smooth, uniform and in good contact with the top of the cell. The signal measured in this area was analysed. The majority of the grains also originated from the top side of the cell surface, despite the evidently three‐dimensional distribution of the label. As determined in test specimens, 80–85% of the grains originated from 3–4 keV electrons. Due to the short range of these electrons, the grains represent the distribution of the label on the part of the cell surface which is in close contact with the emulsion layer. 15–20% of the grains originated from β‐decays with higher energies and add partially to the background. According to these data a determination of the number of receptor sites per unit area of plasma membrane (receptor density) is possible in the surface area of the cell top. Both light microscopy (LM) and SEM were used to analyse autoradiographs. LM analysis is possible in principle but the analytical facilities of SEM are yet superior for the identification of silver grains. The registration of the grains is further facilitated in SEM because of the very large depth of focus.