ELECTRON-MICROSCOPIC AUTORADIOGRAPHY WITH CRYOFIXATION AND DRY-MOUNTING PROCEDURE

The procedures for radioautography of soluble compounds can be divided into two categories, i.e., precipitation wet-mounting radioautography and freeze dry-mounting radioautography. The former procedure is limited in application to only a few compounds, while the latter is universally applicable to any kind of compounds. The cryo-fixation and dry-mounting procedure at the electron microscopic level is described in detail in this article. Freezing is carried out at very low temperatures(-160 degrees C or -196 degrees C) in isopentane cooled with liquid nitrogen or directly in contact with copper blocks cooled with liquid nitrogen. Then the following procedures can be divided into 3 methods, i.e., freeze-dried or freeze-substituted tissues are embedded in Epoxy resin and dry-sectioned, or frozen tissues are directly cryosectioned and then freeze-dried, in order to prevent diffusion artifacts of labeled compounds. Dried thin sections are dry-mounted with emulsions for the radioautographic procedure. Dried films of radioautographic emulsions are applied to the dry sections by means of a wire-loop method, exposed, developed and stained. As the controls, glutaraldehyde and osmium tetroxide fixed, wet-mounted radioautography is employed and compared. As the results, soluble compounds are quantitatively analyzed by subtracting the density of a wet-mounting radioautogram from the dry-mounting one. From the results obtained by the present authors, various soluble compounds, including nucleotide precursors, proteins, lipids, polysaccharides precursors, hormones, drugs and inorganic compounds, are generally localized diffusely in both the nucleus and the cytoplasm of various cells examined. These procedures are expected to be applied in the future for various organic and inorganic substances in living organisms to clarify the sites of their incorporation, synthesis, and discharge.