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At least 19 recordsLinked to original sources
PubMed1979

Whole-body autoradiography of bumetanide-14C in dogs by round saw method.--A combined method of autoradiography and tissue radioassay--.

The distribution of a new potent diuretic, Bumetanide, in dogs was studied by means of a combined method of whole-body autoradiography and tissue radioassay, after oral administration of Bumetanide-14C to five young beagle dogs (ca. 1.2 kg). A round saw method developed by Kalberer was applied to obtain frozen slices of dog whole-body with a thickness of 3 mm. After exposure to X-ray films from both sides of the slices at -60 degrees C, frozen samples of the main tissues as well as the blood, urine, bile and intestinal contents were obtained to be assayed for radioactivity and to be analyzed for metabolites by TLC. The advantages of the round saw method (thick slices) in larger animals as dogs and monkeys as compared to the conventional microtome method (thin sections) were pointed out and discussed.

Administration, Oral↗
PubMed1979

[Electron microscopic autoradiography on semithin sections (author's transl)].

The present study applies semithin sections for electron microscopic autoradiography. Due to (a) the decrease of exposition time, and (b) lower intensity of isotope labelling, low power electron microscopic autoradiography on semithin sections is favoured in comparison with conventional (ultrathin section high power) electron microscopic autoradiography, and due to (c) more precise localization of silver granules, and (d) ease of quantitative evaluation of autoradiography, it is superior to ordinary (thick section) light microscopic autoradiography.

Animals↗
PubMed1992

A new method to localize active renin in tissues by autoradiography: application to dog kidney.

A method was developed to localize active renin in dog kidney sections using autoradiography to detect in vitro binding of the radiolabeled renin inhibitor, 125I-H77. Light fixation by prior perfusion of the kidney with paraformaldehyde was used to immobilize renin without denaturing its binding activity. Snap frozen sections were cut on a cryostat and incubated with 125I-H77. Dry film autoradiography revealed discrete binding of 125I-H77 to the vascular pole of glomerulus as well as diffuse binding to the outer medulla and to the cortex. Binding of 125I-H77 to other aspartyl proteases in the latter two regions was then suppressed by addition of the aspartyl protease inhibitor, N-acetyl-pepstatin (1 microM). This revealed only the juxtaglomerular binding and successfully suppressed binding of the radioligand to other sites. Light microscopic emulsion autoradiography revealed highly selective discrete labelling of the juxtaglomerular apparatus. Competition for this 125I-H77 binding by a series of structurally different renin inhibitors showed a close correspondence between their reported inhibitory potency for renin and potency in the binding system. This strongly suggests that the radioligand binds to the active site of renin immobilized in the kidney. These results demonstrate a new method to localize active renin in tissues using in vitro autoradiography and radioinhibitor binding. The method shows promise for localization and quantitation of tissue renin in extra renal tissues.

Animals↗
PubMed1977

In vivo sterol biosynthesis by pea aphid symbiotes as determined by digitonin and electron microscopic autoradiography.

Pea aphid primary symbiotes have previously been shown to synthesize cholesterol in vitro. Two electron microscopic techniques were used here to determine whether the symbiotes also synthesize cholesterol in vivo and whether this cholesterol is made available to the aphid. We also inquired into a possible role of secondary symbiotes in chosesterol biosynthesis. Treatment of aphids with digitonin resulted in significant alteration of ultrastructural sites in primary and secondary symbiote membranes. We concluded that these sites are areas of high cholesterol concentration in the symbiotes. Electron microscopic autoradiography with 3H-mevalonate precursor indicated that both primary and secondary symbiotes synthesize cholesterol; in both cases, the majority of grains were associated with the symbiote membranes. While the frequency of grains on the symbiotes remained constant, irrespective of incubation time in labelled media, the frequency of grains over surrounding tissues increased exponentially as the time of incubation was increased from 30 min to 8 h, indicating that symbiote cholesterol is transported to other tissues. High voltage electron microscopic autoradiography permitted thick section autoradiography, reducing the time of emulsion exposure from 54 days (thin section) to 12 days (0.5 mum sections).

Animals↗
PubMed1992

Cellular variations in estrogen receptor mRNA translation in the developing brain: evidence from combined [125I]estrogen autoradiography and non-isotopic in situ hybridization histochemistry.

The spatial distribution of cells in the adult rodent forebrain which express estrogen receptor mRNA, as shown by in situ hybridization histochemistry with isotopically-labeled probes, has been reported to overlap with regions that are known targets of estrogen and which bind estrogen. The extent to which detection of estrogen receptor mRNA within developing forebrain neurons of the postnatal day 10-12 female rat is accompanied by translation into estrogen binding sites was investigated by combining [125I]estrogen autoradiography with non-isotopic (digoxigenin) in situ hybridization, using a 48-base oligodeoxyribonucleotide probe encoding a sequence of the estrogen-binding domain of rat uterine estrogen receptor cDNA. Estrogen receptor mRNA and estrogen binding sites appeared to be restricted to neurons. No mRNA or binding was seen in ependymal cells. Cells expressing estrogen receptor mRNA were widely distributed in the developing rat forebrain and were found in brain regions generally corresponding to those previously shown in the adult, with the addition of some regions not previously described, such as the medial habenula and dorsal endopiriform nucleus. Although there was widespread overlapping of estrogen receptor mRNA expression with known estrogen binding sites, there were regional and cellular variations in the extent of receptor mRNA translation. This pattern was true for developing forebrain regions previously defined as estrogen receptor-containing (hypothalamus, preoptic area, medial and lateral septum, vertical and horizontal nuclei of the diagonal band, cerebral cortex, hippocampus and amygdala) as well as for regions heretofore not considered estrogen targets (the thalamus, dorsal endopiriform nucleus, claustrum, ventral pallidum/substantia innominata and the basal nucleus of Meynert) or characterized as estrogen-responsive in the adult without previously documented estrogen binding [caudate-putamen (striatum)]. While estrogen binding and receptor mRNA expression always co-localized, neurons expressing estrogen receptor mRNA did not always exhibit ligand binding and there was no clear-cut relationship between the intensity of the hybridization signal and estrogen binding. Little, however, is known about translational control of estrogen receptor expression in the brain. Localization of estrogen binding sites to regions not generally considered targets of estrogen would appear to reflect the greater sensitivity of the iodinated ligand than the tritiated estrogens more commonly used for autoradiography. Non-isotopic in situ hybridization histochemistry combined with [125I]estrogen autoradiography represents a very powerful tool with which to study regulation of estrogen receptor gene expression at the single cell level with an exceptional degree of cellular and anatomical resolution.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1976

The possibility of electron microscopic autoradiography of steroids after freeze drying of unfixed testes.

A technique has been developed for the autoradiographic localization of steroids in testes at the light and possibly electron microscope level. During processing of the tissue there is no contact of tissue with water up to the moment of photographic development of the autoradiographs. Tritium labelled steroids have been introduced into the testis through perfusion of the isolated organ. Small tissue samples were rapidly frozen, freeze dried and fixed with osmium vapour. The fixed tissue was embedded in Epon and thin sections could be cut from this material on an ultra-cryotome without the aid of floatation liquids. Results with light microscopic autoradiography indicate the potential of this technique. This procedure which avoids any contact of the tissue with water offers good prospects for autoradiography of steroids and other soluble compounds at the electron microscope level. A comparison is made with other proposed techniques for steroid autoradiography.

Androstanes↗
PubMed1979

Freeze-fracture autoradiography. Progress towards a routine technique.

Freeze-fracture autoradiography was introduced in 1976 as a new technique for the autoradiography of diffusible compounds at the electron microscope level. With the original approach coating of the frozen replicated specimens was performed in a cryostat at atmospheric pressure. Ice contamination of the specimen surface acting as an outstanding source of artifacts was thereby not excluded. With the use of a specially designed coating device and volatile spreading substances it was made possible to coat the frozen replicated specimens in the maintained vacuum of the freeze-fracture plant. In this complicated technique we have recently extended the freeze-fracture autoradiography to labeled frozen-dried "half" membranes of red blood cells.

Animals↗
PubMed1977

Electron-microscopic autoradiography of tritiated testosterone in rat testis.

The feasibility of a technique for autoradiography of diffusible substances has been further tested by analysing the localization of steroids in rat testes with the light-and electron-microscope. Testes of rats were perfused with tritiated testosterone (3 min) followed by 15-min perfusion with buffer containing a 100-fold of unlabelled testosterone. Tissue samples were frozen, freeze dried, fixed in osmium vapour and embedded in Epon. To exclude extraction of steroids, contact with water and other solvents was prevented during cutting of thin sections on an ultracryotome and further treatments for autoradiography. Light- and electron-microscopic observations indicate that the highest concentration of labelled testosterone was present within the basal parts of the Sertoli cell cytoplasm and in lipid inclusions of Sertoli cells within the seminiferous tubules. This is the first account of autoradiography of steroids at the electron-microscope level.

Autoradiography↗
PubMed1975

Autoradiography of live and dead mammalian cells with 99mTc-tetracycline.

Considering the diagnostic significance of a few 99mTc-chelates in the diagnosis of myocardial infarct, we have recently developed a necrosis model in tissue culture obtained from mammalian cells, where the dead cells showed enhanced binding of 99mTc-tetracycline like the necrotic cells of infarcted myocardium. Technical details of the principles of labeling, fixing, washing and drying, dipping in emulsion, exposure, development and staining of mammalian cells have been described. Considering the short half-life of 99mTc-radioisotope, the recently available techniques have been modified to make autoradiography possible in a reasonably short period of time. Technetium-99m in different physical and chemical states is playing a very important role in a variety of diagnostic procedures. The autoradiography of 99mTc-labeled cells and tissue will shed further light on the mechanism of cellular uptake and subcellular distribution in normal and pathologic states. Presently no information is available regarding autoradiography with 99mTc-chelates of mammalian cells obtained from tissue culture.

Autoradiography↗
PubMed1976

In vitro labeling and gold activation autoradiography for determination of labeling index and DNA synthesis times of solid tumors.

In vitro labeling and gold activation autoradiography were used to determine the [3H]thymidine ([3H]TdR)-labeling indices and DNA synthesis times for C3H/He spontaneous mammary tumors. Three variations of the [3H]TdR, [14C]thymidine ([14C]TdR) double-labeling method, together with double-emulsion autoradiography, were used to determine the DNA synthesis times (TS). Tumors labeled totally in vivo (in vivo-in vivo method) and tumors labeled with [3H]TdR in vivo and subsequently labeled with [14C]TdR in vitro showed similar TS values. DNA synthesis times for tumors determined totally in vitro by double labeling (in vitro-in vitro method) were significantly longer than those observed in vivo; however, identical samples subjected to Hypaque-Ficoll gradient separation after double labeling showed TS's similar to those found in vivo. Furthermore, the interval between [3H]TdR and [14C]TdR administration had no effect on TS estimates in vitro. Gold activation autoradiography was used in the present experiments to reduce autoradiographic exposure times. This method, together with in vitro labeling, permits [3H]TdR labeling index and TS determinations after 6-hr and 7-day exposures, respectively.

Animals↗
PubMedDate not supplied

Dynamics of steroid uptake in rat testis studied by quantitative autoradiography.

Localization of radioactive steroids in rat testis was studied by autoradiography of tissue section. For autoradiography small tissue samples were frozen, freeze-dried under vacuum, fixed with osmium vapor and embedded in epon. The transfer of radioactive steroids was studied after in vitro perfusion of radioactive steroids in the testes isolated from hypophysectomized animals, thereby excluding the interference of endogenous steroids. Quantitative autoradiography on the basis of grain densities after perfusion of testes with tritiated pregnenolone or testosterone, revealed an accumulation of the label in the Leydig cell cytoplasm. After longer perfusion periods the amount of label in the seminiferous tubules increased and a preferential localization was observed in the basal cytoplasm of Sertoli cells and in lipid droplets. Perfusion of testes with estradiol-17 beta resulted in a distinctly different pattern of radioactivity in the autoradiographs. A high labeling of the Leydig cell nuclei was observed in combination with a low general labeling of all the other cell structures. The results suggest that different steroids are localized in different specific areas of the rat testes in vivo.

Animals↗
PubMed1975

3H-thymidine autoradiography of CSF cells in primary reticulum cell sarcoma of the brain.

CSF cells in a case of primary reticulum cell sarcoma of the brain with diffuse subarachnoid spreading were examined by 3H-thymidine autoradiography. Immediately after lumbar puncture, the CSF withdrawn was incubated at 37 degrees C for 1 hr with an admixture of 3H-thymidine at a rate of 1 muCi/ml CSF. The cells were collected by centrifugation and the microautoradiographic procedure was performed. The labeling index (L.I.) of the total CSF cells was 10.5%, and when non-neoplastic cells, i.e. polymorphonuclear leukocytes, small lymphocytes, monocytes etc., were excluded, the real L.I. of the tumor cells in the CSF was supposed to be more than 14.4%. Referring to the results of various brain tumors reported in the literature, this belongs to the highest labeling group. The high L.I. of the tumor cells in this case was well consistent with the extremely rapid clinical course. It should be stressed that the examination of CSF cells by 3H-thymidine autoradiography in cases of brain tumors could be one of the valuable methods indicating the DNA synthesis of the tumor cells, which is an important parameter of malignancy.

Adult↗
PubMed1977

Phenidone-ascorbic acid development in electronmicroscopic autoradiography.

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.

Albumins↗
PubMed1978

Freeze-fracture autoradiography: the in-vacuo coating technique.

Freeze-fracture autoradiography (FFA) was introduced in 1976 as a new method for electron microscopic autoradiography of diffusible compounds (Fisher and Branton, Rix et al.). With the original technique, the film monolayer was applied to the cold specimen in a cryostat at atmospheric pressure. Coating under these conditions did not exclude the risk of artifacts, mainly due to uncontrolled ice contamination of the cold specimen surface. A new method has been developed for coating the frozen specimen, immediately after replication, in the maintained vacuum of the freeze-fracture unit. Two main components of the new technique are described in detail, a specially designed coating device, and the use of spreading substances, promoting adhesion of the film in vacuo. Using this technique artifacts so far inherent in the FFA method can be eliminated.

Animals↗
PubMed1977

High-speed autoradiography of 3H-thymidine-labelled nuclei.

High-speed autoradiography with stripping film of 3H-thymidine-labelled cells was tested. The tests involved: (a) various times of immersion of emulsion-covered cell preparations in the mixture of dioxane-PPO-POPOP, at 20 degrees C, (b) exposure of cell preparations and blanks for various times at either -70 degrees C or +20 degrees C, with different humidity levels. Autoradiographs of good quality could be produced by 2-min immersion in the scintillator, exposure time greater than or equal to 1 h at either temperature and relative humidity 20--30%. A linear relationship between autoradiographic efficiency and exposure time of 1--7 h was found at either temperature, although the efficiency of autoradiographs exposed at -70 degrees C was by approximately 30% higher than that of autoradiographs exposed at +20 degrees C. Background values of autoradiographs dried with a fan and exposed for 1/4--7 h at either -70 degrees C or +20 degrees C were 0.6--0.8 grain/100 micron2. Theoretical calculations and experimental data showed that high-speed autoradiographs are 30--50 times more efficient as compared with conventional stripping film autoradiographs, thus allowing a shortening of the respective exposure time. Theoretical aspects of efficiency and resolution of high-speed autoradiography are considered.

Autoradiography↗
PubMed1992

Increased binding of 3H-L-deprenyl in spinal cords from patients with amyotrophic lateral sclerosis as demonstrated by autoradiography.

The present investigation has applied quantitative autoradiography and histochemistry to study the regional distribution of MAO-B and its relation to the number of cells in respective regions. L-deprenyl binds irreversibly and quantitatively to the B-form of monoamine oxidase, MAO, and is an ideal 3H-ligand to measure the MAO-B enzyme protein in tissues by means of in vitro autoradiography. The investigation is performed on spinal sections from five controls and five cases with amyotrophic lateral sclerosis (ALS) on cervical, thoracic and lumbar level. The highest density of 3H-L-deprenyl binding was found around the central canal (lamina X). MAO-B was markedly increased (up to 2.5 times of values in controls) specifically in regions of neurodegeneration e.g. motor neuron laminae and corticospinal tracts. There was a high correlation between glial cell count and 3H-L-deprenyl binding with a relation indicating enhanced MAO-B protein in glial cells within areas of neurodegeneration. In contrast the increased microglial cell number in ALS did not show any correlation with 3H-L-deprenyl binding.

Aged↗
PubMed1992

[11C]Ro 15-4513, a ligand for visualization of benzodiazepine receptor binding. Preparation, autoradiography and positron emission tomography.

Ro 15-4513, a partial inverse agonist at the benzodiazepine (BZ) receptor site was labelled with 11C and used for in vitro autoradiography on human post mortem brain sections and for positron emission tomography (PET) on Cynomolgus monkeys. The total radiochemical yield of [11C]Ro 15-4513 was 30-40% with an overall synthesis time of 40 min. The specific radioactivity was about 1000 Ci/mmol at end of synthesis. In vitro autoradiography showed that [11C]Ro 15-4513 bound specifically predominantly in the neocortex of the human brain. Specific binding was also demonstrated in the basal ganglia and the cerebellar cortex. Flumazenil (Ro 15-1788) and clonazepam inhibited the binding in cerebral regions, but a significant proportion in the cerebellum was not inhibited by these agents. This proportion may represent alpha 6-containing BZ receptors. PET examination of [11C]Ro 15-4513 binding in Cynomolgus monkeys demonstrated high uptake of radioactivity in neocortex. The uptake of radioactivity was markedly displaced by high doses of Ro 15-4513 or clonazepam. [11C]Ro 15-4513 should be a useful ligand to examine BZ receptor characteristics in the living human brain by PET.

Animals↗