High-Resolution Autoradiography

Lucien, G.; Tubergen, Robert P. van

doi:10.1083/jcb.15.2.173

Cited by 938 publications

(220 citation statements)

References 18 publications

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“…For the purpose of discussing resolution, we consider Kodak NTE and NTE2 to be synonymous.) The Ilford L4-coated autoradiographs were developed for large grains with Gold-EAS (23) or for small grains with paraphenylenediamine (4,5). The Kodak NTE2 autoradiographs were developed with Dektol (Kodak; diluted: one of Dektol to two of water) at 25~ as previously described (19).…”

Section: Discussionmentioning

confidence: 99%

Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters.

Salpeter¹,

Fertuck²,

Salpeter³

1977

The Journal of Cell Biology

173

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Resolution for 125I-labeled specimens under electron microscope (EM) autoradiographic conditions was assessed experimentally. With this isotope the size of the silver halide crystal was the most important resolution-limiting factor. Heavy metal staining such as is routinely used in preparing animal tissues for EM autoradiography produced an improvement in resolution of -15-20%. For a 500-1,000-/~ biological tissue section fixed with OsO4 and stained with uranyl acetate, we obtained resolution (half distance, HD) values of -800 -120/~ using Ilford L4 emulsion and 500 -70 A using a Kodak NTE-type emulsion. General aspects of resolution-limiting factors and comparison with 3H and 14C values are discussed.On theoretical grounds, 125I is a highly favorable isotope (see Appendix A) for electron microscope (EM) autoradiography. This was early recognized by Kayes et al. (11) in 1962. However, although some calibration studies for both sensitivity and resolution have been performed using this isotope (6, 10), the resolution attainable with this isotope under varying experimental conditions has not yet been systematically assessed. In the present study we have adapted previous calibration procedures used for tritium and ~4C (2,7,20,22) to obtain resolution values for mzsI as a function of section thickness, photographic emulsions, developing procedures, and heayy metal staining as employed in Em autoradiography. The results allowed us to reassess the critical parameters affecting resolution for isotopes of different energy. MATERIALS AND METHODS Resolution SpecimensTwo resolution specimens were used. One was to test resolution in plastic specimens (density 1.1) as a function of section thickness and different emulsion-developer combinations; the second was to test the effect of increasing specimen density by the incorporation of heavy metals such as osmium and uranium, which are used in the fixation and staining of biological material for electron microscopy.RESOLUTION USING PLASTIC TEST SPECI-MENS: The calibration specimen was modeled after that described previously for tritium and 14C (2,20,22) in which a thin film of radioactive polystyrene was sandwiched between a polymerized Epon block and a layer of nonradioactive methacrylate. (The radioactive styrene was thus never exposed to the solvents normally involved in embedding for EM autoradiography, which would

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Section: Discussionmentioning

confidence: 99%

Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters.

Salpeter¹,

Fertuck²,

Salpeter³

1977

The Journal of Cell Biology

173

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“…Grids containing the sections were coated with Ilford L4 emulsion by the method of Caro et al [10] as previously described 0012-186X/79/0017/0379/$01.20 Fig, 1. Thin section of isolated hepatocytes with developed autoradiographic grains (circles).…”

Section: Preparation For Electron Microscopy and Autoradiographymentioning

confidence: 99%

Relationship of binding to internalization of125I-insulin in isolated rat hepatocytes

et al. 1979

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Summary.By quantitative electron microscopic autoradiographic technique, we have previously shown that I2sI-insulin initially localizes to the plasma membrane of isolated rat hepatocytes and is subsequently internalized in a limited region of the peripheral cytoplasm. In the present study, we have shown that when cells are incubated at 20 ~ steady state binding is reached by 60 minutes and maintained up until 120 minutes of incubation while at 37 ~ steady state binding is reached by 10 minutes and maintained for 30 minutes. Under both of these conditions, internalization of the labelled material occurs as a constant function of the binding. These data suggest that under normal conditions the binding of the ligand is an important rate limiting determinant of the internalization process.Key words: Insulin receptor, internalization, endocytosis, hepatocytes, binding, insulin.The initial interaction of 125I-insulin with hepatocytes occurs through specific, saturable receptors on the surface of the plasma membrane. More recently, it has been demonstrated in isolated hepatocytes [1,2,3,4] and in hepatocytes from intact liver [5,6] that the interaction of insulin with its cell surface receptor results in the internalization of the ligand. While initial binding is primarily controlled by the affinity of insulin for the receptor (K) and the number of receptor sites (Ro), the factors controlling internalization are unknown. In the present study, we have investigated the relationship between the binding of the hormone and its internalization. Materials and Methods Cells and ReagentsHepatocytes were isolated from normal 6 to 8 weeks old Wistar rats fed ad libitum using a modification of the method described by Seglen [7]. IasI-insulin was prepared at a specific activity of 250 vCi/gg by a modification of the chloramine-T-method [8]. The labelled insulin was purified by filtration on G-50 Sephadex at 4 ~ prior to each experiment. Incubation ConditionsHepatocytes at a final concentration of 1 • 10 6 eens/rnl were incubated in duplicate in 0.5 ml of modified Krebs Ringer bicarbonate (KRB) (pH 7.7), containing 25 mg/ml bovine serum albumin (Fraction V) and 0.8 mg/ml of bacitracin, with 5 • 10-1~ ~25I-insulin at 20 ~ and 37 ~ for varying periods of time. Bacitracin was added in order to prevent insulin degradation in the medium during exposure to hepatocytes [9]. Identical incubations were carried out in the presence of 2.7 • 10-5mol/1 unlabelled insulin to determine non-specific binding (i. e. cell associated radioactivity in the presence of an excess of unlabelled hormone). At the times indicated, 1 ml of chilled buffer was added to each tube, immediately followed by centrifugation for 20 seconds at 50 • g. The supematant was discarded and the cell pellet quickly resuspended in I ml of chilled buffer and centrifuged for 90 seconds at about 500 • g in a Beckman plastic microfuge tube. Cell pellets were further washed (without resuspension) by chilled buffer containing 100 mg/ml of sucrose. At the end of the wash procedure, 4% g...

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“…Radioautographic Methods.--Ultrathin sections were covered with Ilford L4 emulsion (Ilford Limited, Ilford, England) diluted 1:3 by using the loop method of Caro and van Tubergen (9). Mter an exposure time of 4-6 wk the sections were developed in Microdol X (Eastman Kodak Co., Rochester, N.…”

Section: Methodsmentioning

confidence: 99%

Studies on Antibody-Producing Cells

Gudat

Harris

et al. 1971

The Journal of Experimental Medicine

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A preceding paper described the ultrastructure of antibody-forming cells of mice and rabbits studied in considerable numbers (1). In confirmation of previous studies from this and other laboratories, it was shown that lymphocytes and plasmacytes in various stages of differentiation contributed to the heterogeneity of the antibody-producing cells (2-8). Morphological forms suggesting transition between cells of the lymphocytic and of the plasmacytic series were also described (1). A comparison of cells forming rosettes with those producing plaques showed that the rosette-forming cells (RFC)I were largely in the lymphocytic series, whereas the plaque-forming cells (PFC) were largely plasmacytic (1).For further study of antibody-producing cells, especially in the early stages of proliferation and antibody release, RFC were chosen, since the number of rosettes produced by a given suspension of antibody-producing cells is many times the number of plaques, so that rosette formation would clearly provide the more sensitive technic. In the present study the kinetics of antibody-forming cells was studied both in terms of the numbers of RFC and of uptake of radioactive label. Cells were obtained from antibody-producing spleens of mice and lymph nodes of rabbits, at various intervals after a first or second injection of antigen, for determination of frequency of RFC and of RFC which could be

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High-Resolution Autoradiography

Cited by 938 publications

References 18 publications

Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters.

Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters.

Relationship of binding to internalization of125I-insulin in isolated rat hepatocytes

Studies on Antibody-Producing Cells

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