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

Kopriwa, Beatrix Markus

doi:10.1007/bf00491492

Cited by 127 publications

(53 citation statements)

References 11 publications

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“…After development, sections were floated off in H20 (or eight drops HFI/40 ml H20) (48), picked up with 150 mesh grids, cleared by floating the emulsion side on concentrated acetic acid for < 15 s, stained with 2.5% uranyl acetate for 10 min at room temperature and then lead citrate (49) .Z" each other. Dense labelling was seen primarily under osteoclasts (see arrow in Fig.…”

Section: Methodsmentioning

confidence: 99%

Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure.

Sato¹,

Grasser²,

Endo³

et al. 1991

J. Clin. Invest.

928

541

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Studies of the mode of action of the bisphosphonate alendronate showed that 1 d after the injection of 0.4 mg/kg [3Hjalen-dronate to newborn rats, 72% of the osteoclastic surface, 2% of the bone forming, and 13% of all other surfaces were densely labeled. Silver grains were seen above the osteoclasts and no other cells. 6 d later the label was 600-1,000 itm away from the epiphyseal plate and buried inside the bone, indicating normal growth and matrix deposition on top of alendronate-containing bone. Osteoclasts from adult animals, infused with parathyroid hormone-related peptide (1-34) and treated with 0.4 mg/kg alendronate subcutaneously for 2 d, all lacked ruffled border but not clear zone.In vitro alendronate bound to bone particles with a Kd of -1 mM and a capacity of 100 nmol/mg at pH 7. At pH 3.5 binding was reduced by 50%. Alendronate inhibited bone resorption by isolated chicken or rat osteoclasts when the amount on the bone surface was around 1.3 X 10-3 fmol/Mm2, which would produce a concentration of 0.1-1 mM in the resorption space if 50% were released. At these concentrations membrane leakiness to calcium was observed. These findings suggest that alendronate binds to resorption surfaces, is locally released during acidification, the rise in concentration stops resorption and membrane ruffling, without destroying the osteoclasts. (J.

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Section: Methodsmentioning

confidence: 99%

Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure.

Sato¹,

Grasser²,

Endo³

et al. 1991

J. Clin. Invest.

928

541

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“…The auto radiograms were exposed in lightproof boxes for 2 weeks at 4 °C; controls for both positive and negative chemography were included. The slides were developed in freshly prepared Kodak D-19 diluted 1:1 with distilled water for 5 min, rinsed in distilled water containing 1% (v/v) acetic acid for 1 min, fixed in 20% (w/v) sodium thiosulphate for 7 min, and rinsed in distilled water for 15 min (Kopriwa 1975). Finally, the autoradiograms were air-dried, shielded with a coverslip using a 1:1 mixture of glycerine/gelatine, and photographed with transmitted light-and dark-field illumination.…”

Section: Pituitary Cell Dispersion and G Nrh Receptor Localisationmentioning

confidence: 99%

Gonadotrophs but not somatotrophs carry gonadotrophin-releasing hormone receptors: receptor localisation, intracellular calcium, and gonadotrophin and GH release

Bosma¹,

Kolk²,

Rebers³

et al. 1997

Journal of Endocrinology

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Gonadotrophs are the primary target cells for GnRH in the pituitary. However, during a limited period of neonatal life in the rat, lactotrophs and somatotrophs respond to GnRH as well. Also, in the adults of a number of teleost fishes (e.g. carp, goldfish, and tilapia but not trout), GnRH is a potent GH secretagogue. In studying hypophysiotrophic actions of the two forms of GnRH present in the African catfish (Clarias gariepinus), chicken GnRH-II ([His5,Trp7,TyrK ] GnRH; cGnRH-II) and catfish GnRH ([His5,Asn8]GnRH; cfGnRH), we have investigated the effects of G nRH on catfish gonadotrophs and somato trophs. G nRH binding was examined by incubating dispersed pituitary cells attached to coverslips with 125I~ labelled [D-Argr\T rp 7,LeuH ,Pro9-Net]GnRH (sGnRHa), a salmon G nRH analogue with high affinity for the G nRH receptor. Following fixation and immunohistochemistry using antisera against catfish LH and GH, 125I-labelled sGnRHa was localised autoradiographically and silver grains were quantified on gonadotrophs and somatotrophs. Specific binding of ' I-labelled sGnRHa was restricted to gonadotrophs. Both cfGnRH andcGnRH~II dose-dependently inhibited I-labelled sGnRHa binding to gonadotrophs. To substantiate the localisation of functional G nR H receptors, the effects of cfGnRH and cG nR H -II on the cytosolic free calcium concentration ([Ca ' were examined in Fura-2-loaded somatotrophs and gonadotrophs. GnRH-induced increases in [Ca2+]i appeared to be confined to gonadotrophs, in which both endogenous GnRHs caused a single and transient increase in [Ca2*]}. The amplitude of this [Ca2+]j transient depended on the G nR H dose and correlated well with the G nR H s1 effect on LH release. In vivo experiments demonstrated that G nR H treatments which markedly elevated plasma LH levels had no effect on plasma GH levels, while a dopamine agonist (apomorphine) significantly elevated plasma GH levels. W e con clude that the two endogenous forms of G nR H in the African catfish are not directly involved in the regulation of the release of GH, suggesting that GnRHs cannot be considered as GH secretagogues in teleosts in general.

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“…Following exposure periods, the radioautographs were developed with Kodak D-19B (10). The sections were poststained with uranyl acetate (11) and lead citrate (12) and viewed in the Siemens 1A or 101 electron microscope.…”

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

Cited by 127 publications

References 11 publications

Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure.

Bisphosphonate action. Alendronate localization in rat bone and effects on osteoclast ultrastructure.

Gonadotrophs but not somatotrophs carry gonadotrophin-releasing hormone receptors: receptor localisation, intracellular calcium, and gonadotrophin and GH release

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

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