A reliable, standardized method for ultrastructural electron microscopic radioautography

Kopriwa, Beatrix Markus

doi:10.1007/bf00306855

Cited by 198 publications

(72 citation statements)

References 49 publications

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“…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 Kodak D-19B (10).…”

Section: I-insulin Porcine Insulin (244 Units/mg; Connaughtmentioning

confidence: 99%

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

Bergeron¹,

Levine²,

Sikstrom³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: I-insulin Porcine Insulin (244 Units/mg; Connaughtmentioning

confidence: 99%

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

Bergeron¹,

Levine²,

Sikstrom³

et al. 1977

Proc. Natl. Acad. Sci. U.S.A.

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“…supported on a glass slide or a grid in both light and electron microscope ARG. For emulsion coating, dipping (5,15,19,29), dropping (29,37), wire loop (10,29,31), touching (9,29) and stripping techniques (18,29,34,44) have been used. However, difficulties arise when one tries to apply ARG in SEM observation, because the surfaces of specimens are generally irregular and uneven.…”

Section: Resultsmentioning

confidence: 99%

Three-dimensional observation of the radioactive concanavalin A receptors on the surface of human red blood corpuscles utilizing scanning electron microscope autoradiography.

Suzuki

Mizuhira

1982

Acta Histochem. Cytochem

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“…The procedures for autoradiography used in this study were partially modified from the procedures described by Kopriwa (1973) and Granboulan (1967). Thin sections (60 to 100 rim) were placed on Formvar-coated copper grids, and stained with uranyl acetate and lead citrate.…”

Section: Methodsmentioning

confidence: 99%

Ultrastructural Localization of Cytomegalovirus DNA Synthesis in Infected Guinea-pig Cells

Fong

1982

Journal of General Virology

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SUMMARYUsing electron microscopic autoradiographic techniques, the uptake of [methyl-3H]thymidine into uninfected and guinea-pig cytomegalovirus (GPCMV)-infected guinea-pig embryo (GPE) fibroblast cells was investigated. In GPCMV-infected GPE cells, [methyl-3H]thymidine uptake into cellular DNA was inhibited prior to the onset of virus DNA synthesis as well as during the entire period of virus replication. Virus DNA synthesis increased slowly during the period of active virus replication, which usually lasted from 18 to 48 h post-infection. Only the electron-dense amorphous matrices and fibrillar structures in the nuclear inclusions were associated with the tritiated labels and thus contained virus DNA.

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A reliable, standardized method for ultrastructural electron microscopic radioautography

Cited by 198 publications

References 49 publications

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

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

Three-dimensional observation of the radioactive concanavalin A receptors on the surface of human red blood corpuscles utilizing scanning electron microscope autoradiography.

Ultrastructural Localization of Cytomegalovirus DNA Synthesis in Infected Guinea-pig Cells

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