Electron microscopic radioautographic study of glycoprotein biosynthesis and renewal in the renal glomeruli of the rat

1978 ◽  
Vol 36 (2) ◽  
pp. 476-477
Author(s):  
A. Haddad ◽  
J-J. Lachat ◽  
R. P. Gonçalves
Keyword(s):  
Renal Cortex ◽  
Mesangial Cells ◽  
Electron Microscopic ◽  
Time Intervals ◽  
Light Microscope Level ◽  
Thin Sections ◽  
Main Site ◽  
Renal Glomeruli ◽  
Silver Grains ◽  
Electron Microscopic Radioautography

Biosynthesis, migration and renewal of glycoproteins were studied by radioautography in the renal glomeruli after administering L-3H-fucose to rats which were killed at several time intervals after injection, by perfusion of glutaraldehyde through the abdominal aorta. Small pieces of the kidney were post fixed in 0s04, dehydrated and embedded in Epon 812. Semithin and thin sections of the renal cortex were processed for light and electron microscopic radioautography, respectively. At the light microscope level (Fig. 1) it was observed that the main site of incorporation of 3H-fucose into glycoproteins was the paranuclear region of the visceral epithelial cells (podocytes). Weak paranuclear radioautographic reactions were also observed in endothelial and mesangial cells at 10 minutes after injection. At the longer time intervals these paranuclear reactions disappeared and the silver grains were mostly overlying the several components of the capillary wall. Silver grain counts showed that the peak of the silver grain density over the glomeruli occurred at 4 hours after injection; by 14 days the radioautographic reactions were almost negligible.

scholarly journals LIGHT AND ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF HEPATIC CELL NUCLEOLI IN MICE TREATED WITH ACTINOMYCIN D

1967 ◽  
Vol 33 (3) ◽  
pp. 489-496 ◽  
Author(s):  
J. C. H. de Man ◽  
N. J. A. Noorduyn
Keyword(s):  
Orotic Acid ◽  
Rna Synthesis ◽  
Actinomycin D ◽  
Electron Microscopic ◽  
Granular Component ◽  
Hepatic Cells ◽  
Progressive Loss ◽  
Nucleolar Rna ◽  
Silver Grains ◽  
Electron Microscopic Radioautography

Nucleolar partition induced by actinomycin D was used to demonstrate some aspects of nucleolar RNA synthesis and release in mouse hepatic cells, with light and electron microscopic radioautography. The effect of the drug on RNA synthesis and nucleolar morphology was studied when actinomycin D treatment preceded labeling with tritiated orotic acid. Nucleolar partition, consisting of a segegration into granular and fibrillar parts was visible if a dosage of 25 µg of actinomycin D was used, but nucleolar RNA was still synthesized. After a dosage of 400 µg of actinomycin D, nucleolar RNA synthesis was completely stopped If labeling with tritiated orotic acid preceded treatment with 400 µg of actinomycin D, labeled nucleolar RNA was present 15 min after actinomycin D treatment while high resolution radioautography showed an association of silver grains with the granular component. At 30 min after actinomicyn D treatment all labeling was lost. Since labeling was associated with the granular component the progressive loss of label as a result of actinomycin D treatment indicated a release of nucleolar granules. The correlation between this release and the loss of 28S RNA from actinomycin D treated nucleoli as described in the literature is discussed.

scholarly journals LA SYNTHÈSE DE L'ADN MITOCHONDRIAL CHEZ TETRAHYMENA PYRIFORMIS

1968 ◽  
Vol 39 (2) ◽  
pp. 369-381 ◽  
Author(s):  
Renée Charret ◽  
Jean André
Keyword(s):  
Mitochondrial Dna ◽  
Great Majority ◽  
Tetrahymena Pyriformis ◽  
Logarithmic Phase ◽  
Time Interval ◽  
Electron Microscopic ◽  
Mitochondrial Population ◽  
A Cell ◽  
Silver Grains ◽  
Electron Microscopic Radioautography

Electron microscopic radioautography has been used to study the synthesis of mitochondrial DNA after incorporation of thymidine-3H by cultures in logarithmic phase of Tetrahymena pyriformis during periods ranging from 15 min to 12 hr. The great majority of silver grains are distributed over the macronuclei, the micronuclei, and the mitochondria. The intensity of the label over the entire mitochondrial population is a function of the length of the incubation period within the time interval considered. The intensity of the mitochondrial label was compared with that of the nuclear label. Mitochondria incorporate at the same rate whether the nuclei are synthesizing or not. This persistence of mitochondrial incorporation in the absence of nuclear incorporation excludes the hypothesis of a nuclear origin for mitochondrial DNA. We are not able to determine whether the apparent continuity of synthesis in the entire mitochondrial population of a cell actually represents a series of asynchronous discontinuities.

scholarly journals Appendix: Radioautographic localization of labelled proteins after incubation of guinea-pig islets of Langerhans with [3H]tryptophan

1974 ◽  
Vol 140 (1) ◽  
pp. 22-23 ◽  
Author(s):  
S. L. Howell ◽  
C. Hellerström ◽  
M. Whitfield
Keyword(s):  
B Cells ◽  
Guinea Pig ◽  
B Cell ◽  
Islets Of Langerhans ◽  
Electron Microscopic ◽  
Storage Granules ◽  
Silver Grains ◽  
Electron Microscopic Radioautography ◽  
Significant Concentration

An analysis was made by electron-microscopic radioautography of the distribution of silver grains over storage granules of A2 and B cells after incubation of isolated guinea-pig islets of Langerhans for 17h in the presence of [3H]tryptophan. A significant concentration of labelled proteins was present in the A2 cell, but not in the B-cell granules.

Renal Glomerular Fibrosis in Two Pigs

1995 ◽  
Vol 32 (3) ◽  
pp. 236-241 ◽  
Author(s):  
K. Shirota ◽  
T. Masaki ◽  
H. Kitada ◽  
M. Yanagi ◽  
Y. Ikeda ◽  
...  
Keyword(s):  
Mesangial Cells ◽  
Electron Microscopic Examination ◽  
Collagen Fibrils ◽  
Lymphoid Cells ◽  
Type Iii Collagen ◽  
Electron Microscopic ◽  
Glomerular Lesion ◽  
Deep Blue ◽  
Renal Glomeruli ◽  
Almost All

Massive deposition of collagen fibrils was found in the renal glomeruli of two pigs. The affected pigs were a 6-month-old female hybrid pig with systemic mycobacteriosis and a retired sow showing azotemia. Microscopically, various amounts of a homogeneous eosinophilic substance were deposited within the expanded mesangium of almost all renal glomeruli in both cases. The deposit was also seen occasionally on the glomerular capillary walls in the sow. Capillary lumens were occluded in the glomerular segments with heavy deposition. Obsolescent glomeruli were often surrounded by lymphoid cells. The deposited substance was Congo red negative and stained deep blue with Heidenhain's azan staining. Immunohistochemical evaluation revealed that the major component of the homogeneous substance was type-Ill collagen. Electron microscopic examination showed that the deposits were masses of fibrils of variable length, width, and shape. The fibrils had transverse bands with a periodicity of approximately 60 nm, consistent with collagen fibrils. These glomerular changes were identical to those of collagenofibrotic glomerulonephropathy that has recently been described as a new glomerular disease in humans. The glomerular lesion may be the result of unusual production of type-III collagen by mesangial cells.

scholarly journals Electron microscopic studies of silver-strained proteins in nucleolar organizer regions: location in nucleoli of rat sympathetic neurons during light and dark periods

1981 ◽  
Vol 51 (1) ◽  
pp. 85-94
Author(s):  
M.J. Pebusque ◽  
R. Seite
Keyword(s):  
Dark Period ◽  
Sympathetic Neurons ◽  
Nucleolar Organizer ◽  
Fold Increase ◽  
Staining Procedure ◽  
Nucleolar Organizer Regions ◽  
Electron Microscopic ◽  
Main Site ◽  
Fibrillar Component ◽  
Silver Grains

Ag-AS staining of nucleolar organizer regions was carried out on interphasic superior cervical ganglia neurons of rats sacrificed during light and dark periods. While the Ag-AS technique has mostly been used monolayer cell lines or cell suspensions, the present study showed that in electron microscopy this technique is also applicable to small pieces of tissues. The finest pictures are obtained when (I) all solutions used for the staining procedure are at pH 4.5-4.7 and (2) the second step of the reaction involving ammoniacal silver and formalin developing solutions does not exceed 3 min. The results indicate that in the 2 time periods studied, a positive reaction took place exclusively in nucleolar fibrillar centres and in the fibrillar centres and in the fibrillar ribonucleoprotein (RNP) component (dense fibrillar component). The other nucleolar components, i.e. granular and vacuolar, were devoid of silver deposits. As previously shown in sympathetic neurons, the fibrillar centres of the nucleoli show a 10-fold increase in volume during the dark period. In this period, silver grains were located on both “giant” and small-sized fibrillar centres. The fibrillar RNP component seen either at the periphery of fibrillar centres or in the form of a well-delimited network showed the strongest reaction. The same distribution of silver grains was observed in the sympathetic neurons of rats sacrificed during the light period. Here again, silver accumulation occurred exclusively in the fibrillar centres and the fibrillar RNP component. The same difference in reactivity was observed as for the dark period, the fibrillar RNP component being the main site of the reaction.

scholarly journals THE INFLUENCE OF DEVELOPMENT ON THE NUMBER AND APPEARANCE OF SILVER GRAINS IN ELECTRON MICROSCOPIC RADIOAUTOGRAPHY

1967 ◽  
Vol 15 (9) ◽  
pp. 501-515 ◽  
Author(s):  
BEATRIX MARKUS KOPRIWA
Keyword(s):  
Resolving Power ◽  
Silver Bromide ◽  
Grain Development ◽  
Electron Microscopic ◽  
Duration Of Action ◽  
Small Grains ◽  
Silver Grains ◽  
Electron Microscopic Radioautography ◽  
Selection Of ◽  
Duration Of Development

Under conditions similar to those in electron microscopic radioautography, the development of three different nuclear track emulsions—Ilford L4, Gevaert 307 and Kodak NTE—was investigated by varying the type of developer, duration of action and temperature. These three factors influence: (1) the number of silver grains produced from exposed silver bromide crystals and, therefore, the sensitivity of the emulsion; (2) the production of a few silver grains from silver bromide crystals, unexposed to radiation from the specimen, that is, the background fog; and (3) the size and shape of the developed silver grains, thus influencing the resolving power. The developed silver grains increase in number and in size with increasing duration of development. After complete development in most developers, the silver grains consist of highly convoluted silver filaments and are 2 or 3 times as large as the original silver bromide crystals. However, small grains may be obtained by brief development in Loveland's and p-phenylenediamine developers, in which case only development centers are revealed. Such small grain development improves resolving power, but decreases the number of developed silver grains, thus reducing sensitivity. The most satisfactory emulsion-developer combination under the conditions of the experiment appears to be Gevaert 307 emulsion developed for 2 min in D-19b. Data are also presented to facilitate the selection of the optimal emulsion-developer combination for various experimental situations.

scholarly journals ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF THIN SECTIONS: THE GOLGI ZONE AS A SITE OF PROTEIN CONCENTRATION IN PANCREATIC ACINAR CELLS

1961 ◽  
Vol 10 (1) ◽  
pp. 37-45 ◽  
Author(s):  
Lucien G. Caro
Keyword(s):  
Guinea Pig ◽  
Golgi Complex ◽  
Optical Resolution ◽  
Nuclear Research ◽  
Uranyl Acetate ◽  
Thin Layers ◽  
Pancreatic Acinar Cells ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Electron Microscopic Radioautography

Electron microscopic radioautographs of guinea pig pancreatic exocrine cells were obtained by covering thin sections (∼ 600 A) of OsO4-fixed, methacrylate-embedded tissue with thin layers of Ilford K-5 nuclear research emulsion. After an exposure of 13 days at 4°C., the preparations were photographically processed, stained with uranyl acetate, and examined in an electron microscope. The label used was leucine-H3 injected intravenously 20 minutes before collection of the specimens. Conventional radioautographs of thicker sections (0.4 micron) were also examined in a phase contrast microscope. The advantages obtained from electron microscopic radioautography are: the higher radioautographic resolution (of the order of 0.3 micron) due to the thinness of the emulsion and the specimen, and a high optical resolution permitting a clear identification of the labeled structure. In the guinea pig pancreas this technique demonstrated that, at the time studied, newly synthesized proteins were concentrated in the structures of the Golgi complex and especially in large vacuoles partially filled with a dense material. The vacuoles are probably a precursor to the secretion granules (zymogen granules) in which the label becomes segregated at a later time. These observations demonstrate directly the role of the Golgi complex in the secretion process. They also illustrate the possibilities of this method for radioautography at the intracellular level.

scholarly journals Autoradiographic detection of [125I]-secondary antiserum: a sensitive light and electron microscopic labeling method compatible with peroxidase immunocytochemistry for dual localization of neuronal antigens.

1986 ◽  
Vol 34 (6) ◽  
pp. 707-718 ◽  
Author(s):  
V M Pickel ◽  
J Chan ◽  
T A Milner
Keyword(s):  
Rabbit Antiserum ◽  
Electron Microscopic ◽  
Thin Sections ◽  
Adult Rat ◽  
Dual Localization ◽  
Secondary Antiserum ◽  
Autoradiographic Labeling ◽  
Labeling Method ◽  
Silver Grains ◽  
Autoradiographic Detection

We examined whether autoradiographic localization of [125I]-antirabbit immunoglobulin (IgG) was suitable for light and electron microscopic detection of a rabbit antiserum to the catecholamine-synthesizing enzyme, tyrosine hydroxylase (TH), and whether autoradiographic and peroxidase labeling could be combined for simultaneous immunocytochemical identification of TH and neuropeptides in brain. Adult rat brains were fixed by aortic arch perfusion with acrolein and paraformaldehyde. Vibratome sections of the fixed tissues were incubated with various dilutions of TH antiserum followed by [125I]-secondary IgG. These sections were then directly processed for autoradiography or were incubated with rabbit antiserum to substance P (SP) or methionine [Met5]-enkephalin (ME). These latter sections were then processed by the peroxidase-antiperoxidase (PAP) or conjugated peroxidase methods followed by autoradiography. Exposure periods of 12-20 days for light microscopy or 90 days for electron microscopy yielded substantial accumulations of silver grains even at the highest (1:30,000) dilution of TH antiserum. At this dilution, immunoreactivity for TH was virtually nondetectable by PAP and conjugated peroxidase methods. The differential sensitivities of the autoradiographic versus peroxidase methods provided a means for separable identification of rabbit antiserum to TH and to SP or ME. Ultrastructural analysis of the catecholaminergic neurons in the medial nuclei of the solitary tract (NTS) showed selective cytoplasmic localization of silver grains for [125I]-labeling of TH in perikarya, dendrites, and terminals. Within single thin sections prepared for dual labeling, the peroxidase marker for SP and for ME was differentially localized with respect to autoradiographic labeling of TH.

A simplified method of emulsion coating and development for quantitative electron microscopic radioautography

1978 ◽  
Vol 36 (2) ◽  
pp. 64-65
Author(s):  
K. Yoshida ◽  
F. Murata ◽  
S. Ohno ◽  
T. Nagata
Keyword(s):  
Mass Production ◽  
Identical Condition ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Simplified Method ◽  
Complicated Process ◽  
Critical Problems ◽  
Electron Microscopic Radioautography ◽  
Quantitative Radioautography

IntroductionSeveral methods of mounting emulsion for radioautography at the electron microscopic level have been reported. From the viewpoint of quantitative radioautography, however, there are many critical problems in the procedure to produce radioautographs. For example, it is necessary to apply and develop emulsions in several experimental groups under an identical condition. Moreover, it is necessary to treat a lot of grids at the same time in the dark room for statistical analysis. Since the complicated process and technical difficulties in these procedures are inadequate to conduct a quantitative analysis of many radioautographs at once, many factors may bring about unexpected results. In order to improve these complicated procedures, a simplified dropping method for mass production of radioautographs under an identical condition was previously reported. However, this procedure was not completely satisfactory from the viewpoint of emulsion homogeneity. This paper reports another improved procedure employing wire loops.

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