Localization of glycoproteins in insulin secretory granules by ultrastructural autoradiography.

To determine whether or not the secretory granules of insulin-secreting cells contained glycoproteins, isolated rat pancreatic islets were incubated for 2 and 4 hr in a medium containing L-[3H]-fucose. Quantitative analysis of high-resolution electron microscopic autoradiographs of the insulin-secreting beta cells demonstrated that glycoproteins with fucose residues are contained within the insulin secretory granule.

The use of slotted grids for electron microscopic radioautography.

A method for electron microscopic radioautography on slotted grids is presented which allows examination of the distribution of silver grains over sections of entire structural units without interference by grid bars. Tissue sections of a size such as to fit the opening of the grid slot are placed on slides coated with a Formvar film of sufficient strength to permit transfer of the completed radioautograph onto the grid and to support it over the slot. Sections are block stained prior to radioautography to minimize the risk of loss of the radioautograph during the procedure.

Block-staining tissues with potassium ferrocyanide-reduced osmium tetroxide and lead aspartate for electron microscopic radioautography.

In the hope of devising a method for prestaining tissues en bloc for electron microscopic radioautography, pieces of radioiodine-labeled liver were taken through various combinations of ferrocyanide-reduced osmium tetroxide, lead aspartate, and aqueous uranyl acetate at room temperature or at 60 degrees C. Following the tests, the method adopted for routine use was to block-stain tissues for 2 hr in potassium ferrocyanide-reduced osmium tetroxide at 4 degrees C followed by 1 hr in Walton's lead aspartate at room temperature. This simple method, which requires no manipulation before or after emulsion coating and development of the radioautographs, provides adequate contrast without inducing background fog or artifacts.

An improved method for freeze-fracture radioautography of tissues and cells, as applied to duodenal epithelium and thymic lymphocytes.

An improved method has been devised for the localization of radioactive substances to either one of the leaflets of cellular membranes. After tissue specimens are freeze-fractured and covered with a platinum-carbon replica, they are freeze-dried to allow coating with radioautographic emulsion at room temperature. After exposure at 4 degrees C and development, the emulsion is protected by layers of carbon and grease before the tissue underlying the replica is dissolved in sodium hypochlorite.

Assessment of resolution by half distance values for tritium and radioiodine in electron microscopic radioautographs using Ilford L4 emulsion developed by "solution physical" or D-19b methods.

Half distance values for electron microscopic (EM) radioautographs with the isotopes 3H and 125I were determined using Ilford L4 emulsion processed with either fine grain, solution physical development, or filamentous grain, chemical development with D-19b. 3H- and 125I-line sources, obtained by cutting perpendicular sections from sections of 3H-labeled methacrylate or 125I-labeled thyroid glands, were processed for EM radioautography. The distribution of silver grains around a line source was determined by measuring their distance from the source in photographs of EM radioautographs.

Insulin binding sites localized to nerve terminals in rat median eminence and arcuate nucleus.

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.

Quantitative investigation of scintillator intensification for light and electron microscope radioautography.

Several reports have indicated that scintillators enhance the intensity of radioautographic reactions. If this were the case, the use of scintillators would shorten the exposure time of radioautographs and the production of radioautographs would be accelerated. It was, therefore, decided to examine the effect of scintillators on the intensity of radioautographs in a quantitative manner over uniformly labeled test specimens.

Prolactin binding sites in the rat brain.

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.

Polypeptide hormone binding sites in vivo: initial localization of 125I-labeled insulin to hepatocyte plasmalemma as visualized by electron microscope radioautography.

The rationale of the specific-binding assay was applied to the detection of the liver insulin receptor in vivo. Quantitative electron microscope radioautography indicated that, 3 min after an intraportal injection, 125I-insulin was exclusively located to the hepatocyte plasmalemma.

Influence of emulsion type and atmospheric oxygen on the fading of latent image in electron microscopic radioautography.

The intensity of radioautographic reactions in Ilford L4, Sakura NR-H2 and Kodak NTE emulsions was compared after exposure in either dry air or dry helium gas at 4 degrees C to test the stability of latent images in the presence or absence of oxygen. A light proof container is described in which slides bearing radioactive sections coated with the three emulsions were exposed in dry helium at a constant pressure of approximately 0.5 atm.

A comparison of various procedures for fine grain development in electron microscopic radioautography.

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).

A semiautomatic instrument for the radioautographic coating technique.

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.