Time coating techumique of radlioatntogrcuphy' devised with B#{233}langer iii 1946 (2) oonsists iii covering radioactive sections with fluid enmuulsion.

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Specific binding sites for blood-borne insulin were determined to be selectively localized on axons and axon terminals in the external median eminence and the hypothalamic arcuate nucleus by means of quantitative fine structural radioautography. This localization suggests that discrete populations of hypothalamic nerve terminals are potential targets for the direct effects of insulin and that insulin may act through synaptic mechanisms to influence hypothalamic circuits regulating energy balance and hypophyseal function.

Fine grain development for electron microscopic radioautography was investigated with two types of radioactive specimens: sections of tritiated methacrylate, which provide a homogeneously labeled source for quantitative evaluation of the radioautographic reaction, and sections of 125I-labeled thyroid. Radioautographs were prepared with Ilford L4, Sakura NR-H2, Agfa-Gevaert NUC 307 or Kodak NTE emulsions. The radioautographs were developed with one of several "solution physical" development procedures (Agfa-Gevaert, phenidone-ascorbic acid, p-phenylenediamine developers) or with arrested "direct" developments (D-19b, Elon-ascorbic acid developers). By arresting each development at an early stage of the reaction and at progressively longer time intervals, it was possible to examine the sequence of shapes in the growth of developed silver deposits for each emulsion-development combination. Thus, conditions which resulted in the development of small, round, compact silver deposits were defined for each emulsion. These developments were used in conjuction with gold latensification, a treatment which increases the sensitivity of the emulsions and thus compensates for the lowered sensitivity of fine grain development procedures. The location of the silver deposits in relation to the silver bromide crystals from which they derive was investigated. The emulsion gelatin surrounding the crystals was stained whereas the spaces, which remained after the crystals were dissolved in the photographic fixer, appeared transparent. This analysis permitted the selection of development procedures in which the single or multiple round silver deposits originating from a single crystal will remain within or on the boundary of this crystal. By this method, quantitation of radioautographic reactions composed of small, round silver deposits was studied by using the uniformly labeled 3H-methacrylate sections as a standard source of radiation. The conditions under which grain counting is feasible are discussed.

The principles of the competitive-binding assay were used in conjunction with light microscopic radioautography to demonstrate specific prolactin binding sites localized on ependyma of the rat choroid plexus, a previously unknown prolactin target tissue.

By means of a mechanical coating instrument a fast, simple method to coat specimens with liquid nuclear track emulsion has been devised for quantitative light and electron microscopic radioautography. In both cases, the section is mounted on a glass slide. After the vertically held slide has been immersed in the melted emulsion, the instrument withdraws it at a slow, constant speed. As a result, the specimen is coated with a thin, uniform emulsion layer composed of homogeneously distributed silver bromide crystals. The thickness of the emulsion coat may be standardized by selection of an optimal combination of emulsion dilution, temperature and withdrawal speed.

The rationale of the specific-binding assay was applied to the detection of the liver insulin rece tor in vivo.Quantitative electron microscope radioautogap~y indicated that, 3 min after an intraportal injection,'12I-insulin was exclusively located to the hepatocyte plasmalemma. The specific-binding assay is routinely used in biochemical studies to assess quantitatively the recognition of 125I-labeled insulin (125I-insulin) by its receptor in vitro (e.g., refs. 1-3). Specific binding is based on a consideration of the law of mass action, which states that high concentrations of unlabeled insulin will compete with '25I-insulin for binding to receptor sites. Thus, receptor preparations (experimental samples) are incubated in vitro with saturating or subsaturating (physiologic) levels of 125I-labeled hormone and an identical control preparation incubated with the same amount of l25-Ilabeled hormone but with an "excess" of unlabeled hormone. Specific binding is defined as the difference in bound hormone between the experimental and control samples (1).We have previously applied this rationale to enable the visualization by electron microscope radioautography of polypeptide hormone receptors to purified subcellular fractions derived from the plasmalemma and Golgi apparatus of liver homogenates.t In the present in vivo investigation the approach of the specific-binding assay has enabled us to screen (by light microscope radioautography) several fixation and perfusion methods and has led to the selection of the fixation procedure that most clearly showed specific insulin binding while still maintaining adequate morphologic preservation of the liver tissue. The resulting electron microscope radioautography hence directly visualizes the 125I-insulin and thereby marks the location of the insulin receptor. MATERIALS AND METHODS 125I-Insulin. Porcine insulin (24.4 units/mg; ConnaughtLaboratories, Toronto) was iodinated by chloramine T (4, 5). The 125I-insulin was freshly prepared immediately before each experiment and the integrity of the hormone was assessed by specific binding to microsomes derived from human placenta (4). The specific activity of the 125I-insulin was 160,tCi/,ug. (7). Following dehydration, the blocks were embedded in Epon.The control animals were treated identically except the portal vein injection (0.1 ml) contained 142.5 X 106 dpm of '25I-insulin plus 50 jig of unlabeled insulin.Light and Electron Microscope Radioautography. For light microscope radioautography, semithin (0.5-Mm) sections were prestained in iron/hematoxylin and coated with Kodak NTB2 emulsion (8). Following various times of exposure, the radioautographs were developed with freshly prepared .For electron microscope radioautography, thin sections (silver-grey interference color) were cut on an LKB-Huxley ultramicrotome and a monolayer of Ilford L-4 emulsion was applied (9). Following exposure periods, the radioautographs were developed with . The sections were poststained with uranyl acetate (11) and lead citrate (12) and viewe...