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A Semiautomatic Instrument for the Radioautographic Coating Technique

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Abstract

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.

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... The flat substrate method for electron-microscopical radioautography according to Kopriwa (1967Kopriwa ( , 1973Kopriwa ( , 1975 was used with slight modifications. Serial ultrathin sections (light-gold interference colour, 50-60 nm) were cut and collected on one-sided, frosted, parlodion-coated (0.8% parlodion in iso-amylacetate) slides. ...
... Controls for both negative and positive chemography were included. Ultrathin sections were coated with melted, diluted (1:6 with distilled water) Ilford-L4 emulsion, using a semi-automatic coating instrument (Kopriwa 1967) and dried vertically, to obtain a monolayer of silver bromide crystals. The ultrathin sections from Experiment 1 were exposed in lightproof boxes for 8 days, those from Experiment 2 for 68 days, both at 4~ All sections were processed (1 min) in freshly prepared Kodak D-19 developer (2.0 g metol, 90.0 g Na2SO 3, 8.0 g hydrochinon, 121.0 g NaeCO 3 9 10 HzO, 5.0 g KBr/1 distilled water), rinsed (1 min) in distilled water, fixed (7 min) in 30% sodium thiosulphate and rinsed (15 min) in distilled water. ...
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The possible function of globules and irregular membrane-bound masses in the gonadotropin cells of the pituitary of Clarias gariepinus was studied. Strong secretory stimulation led to the disappearance of the secretory granules from gonadotropin cells but globules and irregular masses remained present. Acid phosphatase was detected enzyme-cytochemically in both globules and irregular masses. Radiolabelling with tritiated amino acids followed by autoradiography demonstrated that globules received radioactive material after secretory granules. The latter received radioactive material within 75 min of administration of radioactive amino acids but globules and irregular masses did not. Although some globules became radioactively labelled within 24 h of the administration of radioactive amino acids, irregular masses remained unlabelled during this period. Secretory granules reacted positively with antisera against and gonadotropin subunits, whereas globules and irregular masses only reacted with the antiserum against the subunit. A moderate anti-7B2 immunoreactivity was demonstrated in secretory granules and globules, whereas irregular masses labelled strongly. The combined cytological results indicate that globules and irregular masses are degradative, possibly crinophagic structures which develop by fusional events from secretory granules to globules and then to irregular masses.
... This 'coating technique' was later refined by Beatrix Kopriwa, an associate of Leblond's who became one of the best specialists in the field. In particular, she devised a semiautomatic coating instrument that made it possible to achieve a constant thickness of the emulsion coat (Kopriwa 1966). This enabled the quantification of autoradiographic reactions by counting the number of silver grains per unit area over reactive histological structures. ...
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In the modern world of cell biology, it is understood that virtually all cells in the body continuously synthesize a multitude ofproteins, and the pathways of synthesis and secretion of these proteins are well established. The continuous turnover of cells in many tissuesis also an accepted idea, and embryonic and adult stem cells are of central importance, both for the cellular economy and in malignant transformation. None of these phenomena were understood at the beginning of the career of Charles Philippe Leblond, and his extraordinary contributions to these fields have fundamen–tally changed our concepts of cell biology.
... For EMA, sections of the radioactive sources (see below) were transferred to collodion-coated glass slides and covered with a thin layer of carbon. The slides were covered with a slightly overlapping layer of silver bromide crystals of Ilford L4 emulsion, using a dipping apparatus as designed by Kopriwa (1967a). The emulsion was diluted 1:2,5 with demineralized water. ...
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Phenidone-ascorbic acid development in electronmicroscopic autoradiography, using Ilford L4 as photographic emulsion and microdol-x as reference developer. Grain yield and efficiency were studied on pale gold section of uniformly labeled tritium methacrylate. For determination of the resolution, a radioactive line source was prepared by cross-sectioning of an epon-embedded film of tritium labeled albumin. The spatial relationship between silver grains and silver bromide crystals was investigated by shadowing the emulsion with platinumcarbon before development. In shadowed autoradiographs both, silver grains and silver bromide crystal were visible. Phenidone was about twice as sensitive as microdol-x and had a half distance value (Salpeter et al., 1969) of 175 mm. Most of the silver grains of both developers were located within the perimeters of their parent silver bromide crystals. In the case of phenidone more than 80% of the excited crystals gave rise to just one silver deposit. These parameters, together with grain size and shape, and counting feasibility make phenidone a useful developer for quantitative EM-autoradiography.
... Semithin (0 .5 M) Epon sections were radioautographed (31) using an exposure of 4 wk after tyrosine-3H and 10 days after galactose-3 H injection ; and silver grains were enumerated per unit area (Tables I and II) . Grey-to-silver sections were radioautographed for electron microscopy (32)(33)(34)(35), in which case exposure was for 7 months after tyrosine-3H and 2 months after galactose-3 H injection . Radioautographs were developed in Elon-ascorbic acid with previous gold latensification for 5-6 min at 24 âC. ...
... Sections were stained with uranyl acetate and lead citrate (Gupta et al. 1966), dried and covered with a thin layer of evaporated carbon (Salpeter & Bachmann, 1964). Slides were then coated with a tightly packed monolayer of either Ilford L4 (purple interference colour) or Agfa-Gevaert NUC 307 (pale gold interference colour) nuclear track emulsions, using a Kopriwa-type semi-automatic coating machine (Kopriwa, 1967a). Under our laboratory conditions, the dilution needed was 2:3 for Ilford L4 and 3:2 for Gevaert NUC 307; both emulsions being used at 35 0 C and with a withdrawal speed on the machine of 48 mm./min. ...
... Cross sections of the bones were made at approximately I-µm thickness and were stained with azure 11-methylene blue for routine examination . A series of unstained sections from each set of bones from each experiment were mounted on glass slides and subsequently coated with a dipping machine (25) with Eastman Kodak NTB-l I emulsion (Eastman Kodak Co ., Rochester, N .Y .) . Slides were developed in Dektol after being exposed for 12 wk, fixed in Kodak acid fixer, and then stained with azure II-methylene blue . ...
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Embryonic chick cranial bone was cultured in the presence of the antimicrotubular agents, colchicine and vinblastine, and with a number of other compounds known from previous studies to affect the cellular handling of collagen. Secretion of procollagen, quantitated by light microscope autoradiography, was correlated with the extent of conversion of procollagen to collagen and with rates of collagen and noncollagen-protein synthesis. Colchicine inhibited procollagen secretion and conversion to collagen and specifically inhibited collagen synthesis. Cells exposed to colchicine revealed an increased number of dilated Golgi-associated vacuoles and vesicles, some of which contained parallel aggregates of filamentous structures. These observations suggest that the pathway of at least a fraction of procollagen secretion by osteoblasts includes the Golgi complex. Disruption of microtubules may interfere with the movement of Golgi-derived vesicles, and the resulting accumulation of collagen precursors in the Golgi complex may lead secondarily to an inhibition of synthesis. Although vinblastine also inhibited both procollagen secretion and conversion to collagen, the observed reduction in general protein synthesis and striking changes in the ultrastructure of the rough endoplasmic reticulum complicated interpretation of the effects. Interpretation of the effects of cytochalasin B was limited by the finding that the cellular response in cranial bone was markedly heterogeneous and that, contrary to some previous reports, the drug caused an inhibition in the incorporation of radiolabeled amino acids into both collagen and noncollagen protein.
... Semithin (0 .5 M) Epon sections were radioautographed (31) using an exposure of 4 wk after tyrosine-3H and 10 days after galactose-3 H injection ; and silver grains were enumerated per unit area (Tables I and II) . Grey-to-silver sections were radioautographed for electron microscopy (32)(33)(34)(35), in which case exposure was for 7 months after tyrosine-3H and 2 months after galactose-3 H injection . Radioautographs were developed in Elon-ascorbic acid with previous gold latensification for 5-6 min at 24 âC. ...
Article
The parathyroid glands of young rats were radioautographed after a single injection of the protein precursor tyrosine-³H in the hope of identifying the sites of synthesis and migration of newly formed protein in the gland cells. The same procedure was used after injection of the glycoprotein precursor galactose-³H. As early as 2 min after intravenous injection of tyrosine-³H, the label was mainly found in the rough endoplasmic reticulum suggesting that cisternal ribosomes are sites of protein synthesis. By 5 and 10 min, much of the label had migrated from the rough endoplasmic reticulum into the Golgi apparatus. By 20 and 30 min, some label had migrated from there into secretory granules. By 45 min and 1 hr, the label content of the cell had decreased, indicating release of labeled material outside the cell. At 2 min after intravenous injection of galactose-³H, the label was mainly present in the Golgi apparatus, where presumably galactose is taken up into glycoprotein. By 10 min, some label appeared in secretion granules and by 30 min release of the material to the outside of the cell was under way. In conclusion, it is likely that the tyrosine-labeled protein material consists mainly of the parathyroid hormone. The galactose-labeled carbohydrate material would be either associated with the hormone in the cell or be part of a distinct glycoprotein which may be the one present on the outer surface of the plasma membrane (cell coat).
... fined by Rogers (30,31) were modified and standardized for our material (5) Liquid Ilford L4 nuclear track emulsion (Ilford Ltd., Ilford, Essex, Engi,md) was mixed 1:1 by weight with 1% glycerol at 42~ Unstained sections affixed to thoroughly precleaned slides were dipped into emulsion, using a simplified Kopriwa dipping machine (20) at a constant rate of 10 cm/min. Safelight conditions (OC Wratten filter [Eastman Kodak Co.] and 7.5-W bulb) were maintained throughout, and coated slides were air-dried at room temperature in 40-50% humidity for at least 1 h. ...
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Rat thyroid lobes incubated with mannose-3H, galactose-3H, or leucine-3H, were studied by radioautography. With leucine-3H and mannose-3H, the grain reaction observed in the light microscope is distributed diffusely over the cells at 5 min, with no reaction over the colloid. Later, the grains are concentrated towards the apex, and colloid reactions begin to appear by 2 hr. With galactose-3H, the reaction at 5 min is again restricted to the cells but it consists of clumped grains next to the nucleus. Soon after, grains are concentrated at the cell apex and colloid reactions appear in some follicles as early as 30 min. Puromycin almost totally inhibits incorporation of leucine-3H and mannose-3H, but has no detectable effect on galactose-3H incorporation during the 1st hr. Quantitation of electron microscope radioautographs shows that mannose-3H label localizes initially in the rough endoplasmic reticulum, and by 1–2 hr much of this reaction is transferred to the Golgi apparatus. At 3 hr and subsequently, significant reactions are present over apical vesicles and colloid, while the Golgi reaction declines. Label associated with galactose-3H localizes initially in the Golgi apparatus and rapidly transfers to the apical vesicles, and then to the colloid. These findings indicate that mannose incorporation into thyroglobulin precursors occurs within the rough endoplasmic reticulum; these precursors then migrate to the Golgi apparatus, where galactose incorporation takes place. The glycoprotein thus formed migrates via the apical vesicles to the colloid.
Chapter
Material elsewhere in this volume provides ample documentation of the existence of specific receptor molecules for a variety of signal-transmitting molecules acting on cells. The neurotransmitters and the circulating hormones are the most obvious categories of these messengers (although far from the only ones). We shall use the term receptor here to denote such structures in the cell plasma membrane, equipped specifically to recognize primary messenger molecules arriving at their cell and to trigger directly the cellular response to these or to initiate the required signal to other machinery that generates the response (e.g., a separate nucleotide cyclase). In contrast, messenger-binding components in the cell interior which are not concerned with the primary recognition of externally incident messengers, while they may be involved in some cases in mediating the cellular response (e.g., to thyroid or steroid hormones), present an entirely different aspect to the problem of receptor location and will not be considered here. Receptors involved in the immune response, which are a very special case, are also outside the scope of this chapter.
Article
Recent studies, reviewed by Grant and Prockop, have provided evidence for an intracellular form of collagen referred to as "procollagen". This molecule is similar to the rod-like tropocollagen molecule except for the presence of a 13nm terminal piece. The conversion of procollagen to tropocollagen by cleavage of the terminal piece is believed to take place extracellularly. Fine structural studies on odontoblasts, the cells which secrete the collagen of dentin matrix, indicate that filamentous structures, interpreted as procollagen, are condensed into secretory granules in the Golgi apparatus, transported to their sites of secretion in the odontoblast process and released to the cell surface by exocytosis. To determine whether similar events occur in osteoblasts, the elaboration of collagen by these cells was investigated using radioautography and electron microscopy.
Chapter
This chapter discusses the comparison of a new method with usual methods for preparing monolayers in electron microscopy autoradiography. At present two techniques are used most frequently to prepare monolayers of nuclear emulsion for electron microscope autoradiography. The use of a mechanical device for dipping the slides into the emulsion allows the highest standardization of the process. The method is simple and quick, although a loss of specimens may occur when they are taken off the slides and placed on the grids. Monolayers obtained in this way are uniform and highly reproducible. The monolayers are selected according to the interference color. The glossy side of grids rinsed in carbon tetrachloride is placed on the floating film. The grids are then collected by means of a moist filter paper applied over a porous funnel connected with a water pump aspirator.
Article
Axenic trophozoites of Entamoeba histolytica showed increased logarithmic growth but absence of "chromatoid" material (stacked helical arrays of ribonucleoprotein [RNP]) when grown in an all-liquid monophasic culture. Organisms grown in a liquid overlay on a semisolid slant (biphasic medium) showed slow logarithmic growth and the presence of chromatoid material. Chromatoid material accumulated in the rapidly growing trophozoites from monophasic culture during treatment with the Vinca alkaloid, vinblastine. Many of the glycogen-free regions of vinblastine-treated trophozoites as well as, to a lesser degree, of normal cells grown in monophasic and biphasic cultures, contained free ribosomes and randomly oriented 60 A filaments. As ribonucleoprotein assumed the packed helical configuration, areas consisting of parallel, packed filaments could be detected adjacent to and continuous with the ordered RNP arrays. This arrangement could be visualized most frequently in vinblastine-treated trophozoites grown in monophasic cultures. Depending on the tilt of the section with respect to the longitudinal axis of individual helices, 60 A filamentous material could be demonstrated associated with the RNP helices. Localization of ribonucleoprotein precursors was followed by means of high resolution radioautography with uridine-³H and cytidine-³H. With a short (30-min) pulse, label could be visualized only over the glycogen-free areas containing free ribosomes and filaments. With 60-min pulses, label could also be seen over the packed helical arrays. With 30-min pulses followed by a 60-min cold chase, label was seen chiefly over RNP helices. It is postulated that the areas containing ribosomes and filaments represent sites of assembly of the RNP helices possibly on a filament protein column. The possibility that the final helical configuration may be due to a property of this protein is suggested.
Article
A quantitative ultrastructural radioautographic study of in vitro protein synthesis has been carried out in rat synaptosomal fractions incubated with tritiated leucine or a tritiated amino acid mixture. Analysis of grain density distribution demonstrated that presynaptic endings are labeled. 30–50% of the developed grains, representing tritiated amino acids incorporated into proteins, were related to presynaptic endings which accounted for 75–77% of the total processes. 34–45% of the grains were related to processes containing ribosomes which accounted for only 4–7% of the total processes. The relative specific activity of these ribosome-containing processes, some of which could be identified as postsynaptic elements, was up to ten times higher than that of the presynaptic ending. These findings indicate that protein synthesis takes place in vitro in presynaptic terminals although to a significantly lesser degree than that occurring in ribosome-containing processes, which, with other nonpresynaptic processes, are at the present time unavoidable contaminants of synaptosomal fractions. Presynaptic endings that in radioautographs contained no mitochondria were labeled. Also, presynaptic endings were labeled after incubation in the presence of chloramphenical which inhibited 20% of the protein synthesis of the synaptosomal fraction. It is concluded that besides mitochondrial protein synthesis, another protein synthesizing system operates in presynaptic endings in vitro.
Article
An attempt was made to elucidate the cytological nature of the defense mechanisms of the normal middle ear with special emphasis on the secretory system. Autoradiography was performed with the use of isotope-labeled glucose in order to determine the rate of glucose uptake by middle ear cells, particularly by secretory cells. Mucus-secreting glands in the guinea pig eustachian tube incorporated a large amount of glucose, while the demilune cells (serous) incorporated only a moderate amount. Some simple squamous epithelium of the bulla also took up a large amount of glucose. Since the mucous blanket of the bulla was labeled after only one hour, it is speculated that these flat cells are of the secretory type. A cytochemical demonstration of the enzyme acid phosphatase was done in order to determine the localization of this lysosomal enzyme in the middle ear cells. A strong reaction was consistently noted in the cytoplasm of both light (mucous) and dark (serous) secretory cells. This reaction appears to take place on the outer surface of the secretory granules, but, occasionally, it takes place inside the granules. The strongest reaction was noted in the lysosomal bodies. A weak reaction was frequently noted in the nuclear chromatin of the secretory cells. An immunofluorescent study demonstrated the presence of immunoglobulin-producing cells in the eustachian tube and in the guinea pig middle ear mucosa. Fluorescein-labeled cells (round cells) were numerous, particularly in the eustachian tube near the glands. It can be suggested from the foregoing that the middle ear is provided with mucociliary, enzymatic, and immunological (secretory and serum) defense systems. The pathological characteristics of human serous otitis were also reviewed. Evidence was shown of an increase in the number of secretory and ciliated cells, of free macrophages, and of the round cells of the submucosal connective tissue. Evidence of the free escape of transudate through intercellular spaces of the epithelial cells and capillary wall damage in the submucosal layer was also noted in pure serous effusion cases. These changes are identical to those that take place in inflammatory reactions. The middle ear effusion, therefore, can be looked upon as the result of enhanced middle ear defense mechanisms occurring in different phases.
Article
Sub-mottling and mottling doses of fluoride are known to alter the uptake of amino acids by young secretory ameloblasts in the rat. Within the sensitivity of the autoradiographic methods used, these fluoride doses did not alter the rate of synthesis of RNA by ameloblasts, using [3H]-orotate as a precursor.
Article
These studies have examined the ability of smooth muscle cells from developing aorta of the prepubertal rat to utilize amino acids in the synthesis and secretion of connective tissue proteins. Prepubertal rats, previously given either an alcohol carrier or estradiol-17-beta, were each given an intravenous injection of proline-3H. The animals were sacrificed after 15 and 30 min, and 4 hr. Light and electron microscope radioautographs of the aortic smooth muscle and of the myometrial cells demonstrated that the aortic cells, in both groups of animals, and the myometrial cells, in the estrogen-stimulated animals, took up the proline and rapidly secreted it in both collagen and elastic fibers within 4 hr. In contrast, the myometrial cells of the nonstimulated animal took up relatively small amounts of proline and retained most of the amino acid within the cells. Electron microscope radioautographs demonstrated that the organelles involved in this activity were the rough endoplasmic reticulum and Golgi complex together with peripheral elements, presumed to be small vesicles. These studies have demonstrated that the smooth muscle cells of the developing aorta and of the estrogen-stimulated myometrium have a capacity to synthesize and secrete proteins associated with the extracellular connective tissue matrix.
Article
A model system is described for the study of capture reactions for diffusable compounds in enzyme cytochemistry. The model, which allows the investigation of the influence of the composition of the cytochemical medium, the enzymatic activity, and the dimensions of the enzymatic site on the capture reaction, consists of very thin homogeneous layers of enzyme (0.01–0.1 m thick) on glass, which are incubated in the cytochemical medium. The fraction of the total amount of liberated product precipitated in the enzyme layer is dependent not only on the trapping efficiency of the cytochemical medium but also on the concentration of the primary reaction product that can be built up in the enzyme layer. Calculations were performed to determine the steady-state concentration of the primary reaction product that can be built up in the enzyme layer. Acid phosphatase was used as enzyme. The problems associated with the model and its applicability to other types of cytochemical reactions are discussed.
Article
Autoradiographs obtained from 13-day chick sternal chondrocytes indicate that the incorporation of 3H-proline into collagen, and of Na235SO4 into chondroitin sulfates, are concurrent. Matrix products produced by these cells were identified by their susceptibility to degradation by collagenase and testicular hyaluronidase. Incorporation of the precursors was time dependent, and it was blocked by the addition of puromycin to the culture medium. The labeling patterns of individual chondrocytes show that synthesis of matrix components differs from cell to cell within the sternal population.
Article
The apparatus described is designed for monolayer emulsion coating in electron microscope radioautography. The slides with the sections are not withdrawn from the emulsion, but it is allowed to flow out of the dipping jar thus coating them.
Article
Pieces of integument containing cuticle and hypodermal cells from the desert locust Schistocerca gregaria were pulse-labelled to produce very narrow bands of radioactive chitin or resilin in the cuticle. Araldite sections of such cuticle were used as a radioactive line source to test the resolution and the possible effect of self-absorption due to heavy metal staining of tissue-sections in EM autoradiographs. The approximate position of the source and the half-distance of resolution (HD) in EM autoradiographs were determined by replotting the grain-distribution in integrated form, producing a sigmoid-shaped curve. The values of HD, the position of the source and the total number of grains were then used to superimpose a theoretical grain-density distribution function on the experimental grain-distribution histograms. The results confirm the line or very narrow band nature of the radioactive chitin or resilin in the tissue-sections used. When the source was resilin situated in the cuticle at about 1 μm from the cell-surface, the values for HD were 130–180 nm in Ilford L4 and about 100 nm in Agfa-Gevaert NUC 307. When the source was chitin situated just outside the cell surface, the corresponding value for HD in Ilford L4 was 85–125 nm. Possible adverse effects of electron beam damage on resolution in resilin-containing samples are discussed. In sections with radioactive chitin at the cell surface, the total number of grains in the cell-side of the histogram was always significantly lower than that in the cuticle-side. This marked asymmetry in grain distribution is considered to be indicative of much greater self absorption of beta-particles from the tritium source, in heavily stained areas of the sections.
Article
SUMMARYA semi-automatic device is described for the coating of slides with nuclear emulsions for autoradiography. The design embodies a metal heatsink for warming the emulsion, and an elevating gantry which withdraws coated slides at a controlled constant speed.
Article
The applicability of tracheal organ culture as an experimental model for the study of disease mechanisms of many different species of mycoplasmas has been demonstrated. Techniques employed using this sytem to study the pathogenesis of M. pneumoniae disease included: stroboscopic quantitation of ciliary activity: light, immunofluorescence and electron microscopy: liquid scintillation spectrometry: and radioautography. The organ culture model and the techniques described should be of value with modification for use with tissue from other organs and animal species for the study of microbial agents that attack these structures in natural disease.
The effects of vitamin A-deprivation on the tracheal epithelium were studied in 35-day old hamsters that had been raised since birth on a vitamin A-deficient diet. Colchicine and3HTdR were given 6 hours before death and the proliferative activities of basal cells and mucous cells were quantified separately by3HTdR labeling indices and mitotic rates. At foci of stratification and epidermoid metaplasia, cell replication rates were increased over controls and more than 70% of all mitotic activity was associated with “non-basal” cells. Genesis of these lesions The opinions and assertions contained herein are the private views of the authors and are not to be construed official or as reflecting the views of the Department of Defense The experiments reported herein were conducted according to the principles set forth in the “Guide for the Care and Use of Laboratory Animals” Institute of Laboratory Animal Resources, National Research Council, DHSW Publ. No. 78-23 This is contribution No. 1554 from the Cellular Pathobiology Laboratory of the Department of Pathology, University of Maryland School of Medicine was coincident with cell death and cell loss. The histogenesis of stratification and epidermoid metaplasia was characterized. Morphological evidence indicated that these lesions were closely related histogenetically and were composed, for the most part, of altered mucous cells which expressed dual phenotypes i.e. keratinization and mucus synthesis.
Article
In intact male rats after TRH administration for 7 and 14 days, TSH cells showed similar morphological changes to those observed after thyroidectomy. These changes were paralleled by small numerical increases in TSH cell counts. After 34 days of TRH treatment, however, most of the TSH cells had a normal appearance and the number of TSH cells also had returned to normal. TRH treatment for 7, 14 and 34 days caused morphological changes in Prolactin cells similar to those obtained after a suckling stimulus. In the three groups these changes were also paralleled by small numerical increases in Prolactin cell counts. The cell replication after TRH for 7 and 14 days, as measured by incorporation of tritiated thymidine to obtain a labeling index, was slightly but significantly increased.
Chapter
Light microscope radioautography (1–12) is a technique that involves the administration of a radiolabeled compound of suitable specific activity (see radioisotopes dose and route of administration) to cells, tissues in culture, or even whole tissues followed by the preparation of sections from paraffin/paraplast or resin-embedded specimens (see Chapter 2). The sections are coated with the appropriate fine grain, photographic emulsion (see Table 1) and the section—emulsion sandwich is incubated in a light-tight container for various times. Then, the section—emulsion sandwich is developed with photographic solutions appropriate for the selected photographic emulsion. Subsequent to development, the task of grain quantitation can be laborious. Such methods as manual grain counting have been performed using a hemocytometer or observing the image Emulsion type Required safelight Developmentb Kodak NTB-2 and NTB-3 nuclear track emulsion (store up to 2 mo at 4°C) Kodak safelight filter no. 2 using 15-W bulb 4 ft from working area Kodak Dektol developer or Kodak developer 19 followed by washing and fixing in Kodak fixer— followed by washing NTB-2 (less sensitive than NTB-3 and useful for [3H], [1251], [14C], and [35S] Amersham LM-1 See Amersham Technical Bulletin Kodak AR-10 stripping film (store up to 6 mo at 4°C in its shipping container) Bulletin Kodak safelight filter no. 1 (red) Kodak Dektol developer or D-19 Ilford G5, KO, Kl, K2, K5 and L4, G5, and L4, G5, and K5 differ in crystal size but both have a high level of sensitivity; K2 is useful for [3H] and [125I] See Chapter 17, electron microscope D-19, Microdol X with a dark-field microscope while illuminating the grains as white specks; the remainder of the tissue is black. Advances in image analysis software have eliminated human counting error; however, the task is still rather labor intensive.
Article
Phenidone-ascorbic acid development was calibrated for electronmicroscopic autoradiography, using Ilford L4 as photographic emulsion and microdol-x as reference developer. Grain yield and efficiency were studied on pale gold sections of uniformly labeled tritium methacrylate. For determination of the resolution, a radioactive line source was prepared by crosssectioning of an epon-embedded film of tritium labeled albumin. The spatial relationship between silver grains and silver bromide crystals was investigated by shadowing the emulsion with platinumcarbon before development. In shadowed autoradiographs both, silver grains and silver bromide crystal were visible. Phenidone was about twice as sensitive as microdol-x and had a half distance value (Salpeter et al., 1969) of 175 mm. Most of the silver grains of both developers were located within the perimeters of their parent silver bromide crystals. In the case of phenidone more than 80% of the excited crystals gave rise to just one silver deposit. These parameters, together with grain size and shape, and counting feasibility make phenidone a useful developer for quantitative EM-autoradiography.
Article
Five commonly used developers (Acufine, Rodinal, Dektol, D19 and Microdol X) and two popular nuclear gel emulsions (Kodak NTB-2 and Ilford L4) were tested in combinations for adequacy of development characteristics and the distribution of silver particle (grain) size measured by means of a reflectance microphotometer. All developer-emulsion combinations had some range of development time during which maximum development was accomplished with no occurrence of background grains, with the exceptions of the combinations of NTB2-Acufine and NTB2-D19. Ilford L4 emulsion obtained the narrowest grain size distribution with Rodinal and Acufine, followed by Dektol, and D19. The narrowest size distribution for Kodak NTB2 emulsion was achieved with D19 followed by Acufine, Dektol and Rodinal. Microdol-X had undesirable effects on the integrity of the individual grains of both emulsions. The criteria are discussed for selecting the most advantageous emulsion-developer system for the particular mode of evaluation (visual, photometric, television camera) to be applied to the finished autoradiographic specimens.
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