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. The grease is removed in Freon 14 and the replica with its emulsion cover is mounted on a specimen grid for electron microscopic examination. The accuracy of radioactivity localization was demonstrated using 3H-thymidine-labeled liver by finding silver grains over the same sites after freeze-fracture as after thin section radioautography. Tests with 3H-methacrylate revealed that the interposition of a platinum-carbon replica decreased the radioautographic reaction by over 80%; hence, the need for long exposure. Only 67% of the silver grains came from radiation sources located beyond the upper 0.05 micron of the specimen and, therefore, the emulsion could be affected by radiation sources located not only within membrane leaflets but also in nearby cytoplasm. Thus, when 3H-fucose was injected into rats to locate newly formed glycoproteins within intestinal epithelium membranes, some of the silver grains found over E and P faces might be produced by radiation coming from the adjacent cytoplasm. To localize label within membrane leaflets in the absence of radiation sources in the cytoplasm, lymphocyte suspensions were incubated with 3H-concanavalin A at 0 degrees C. The plasmalemma radioactivity was then restricted to the two membrane leaflets, with 87-93% of the silver grains on the E leaflet and 7-13% on the P leaflet. It appears that, under these conditions, the technique provides adequate localization of radioactivity to the leaflets of the cell membrane.

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. The number of silver grains per unit distance from the line source was plotted on graphs and half distance values were calculated. With solution physical development, the half distance value was 74 nm for 3H and 80 nm for 125I; whereas with D-19 b development it was 187 nm for 3H and 157 nm for 125I. Since solution physical development produced a reduction of about 50% in the half distance values for both isotopes, it is concluded that the production of fine grain by this method provides better resolution for EM radioautography than filamentous grain development with D-19b.

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. Reaction intensities with or without scintillators were compared using Kodak NTB2 emulsion for light microscopy and Ilford L4 and Sakura NR-H2 emulsion for electron microscopy. In addition, the background fog was quantitatively evaluated. No increase in the intensity of the radioautographs was observed under the following conditions: pretreatment of tissue sections with scintillator solutions before emulsion coating and exposure; treatment with scintillator solutions after emulsion coating but before exposure; treatment with scintillator solutions during exposure; contact with a plastic scintillator film throughout exposure. Heightened reaction intensity was obtained only when scintillators were applied during the histological processing of tissues. The impregnation of tissue blocks with PPO (2,5-Diphenyloxazole) and POPOP (1,4-bis-2-(5-phenyloxazolyl)-benzene) during dehydration, infiltration and Epon embedding produced a 20% increase of reaction intensity in light microscope radioautographs using NTB2 emulsion and a 75% increase in electron microscope radioautographs using L4 emulsion.

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.

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. The comparison of air and helium atmospheres during exposure of radioautographs was estimated qualitatively for 125I-labeled thyroid sections stored for several years and, in addition, quantitative data was derived from 3H-labeled methacrylate sections stored from 21 days to 1 year. With the three emulsions under study, the background fog remains low under both exposure conditions at 4 degrees C for as long as several years duration. Using L4 emulsion, similar high grain densities are obtained in air and helium, and therefore, the latent images in L4 emulsion remain stable in the presence of oxygen. In the case of NTE and NR-H2 emulsions, as the exposure time increases, substantially lower reaction intensities are observed in air than in helium. This difference in reaction intensity is evident by 3 weeks with NTE and after 4 weeks with NR-H2. Hence, there is fading of the latent images in the latter emulsions in the presence of oxygen. It is concluded that reliable results may be obtained with the L4 emulsion by exposure of radioautographs in dry air, whereas with the NR-H2 and NTE emulsions, exposure should be in an oxygen-free medium, such as is provided by a dry helium atmosphere.

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. 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.