Comparison of Cholesterol Extraction from Tissues During Processing for Electron Microscopic Radioautography

1968 ◽  
Vol 26 ◽  
pp. 92-93
Author(s):  
E. Tessa Hedley-Whyte ◽  
Betty G. Uzman
Keyword(s):  
Electron Microscope ◽  
Lipid Metabolism ◽  
Propylene Oxide ◽  
Mouse Liver ◽  
Uranyl Acetate ◽  
Electron Microscopic ◽  
Electron Microscopic Radioautography

Radioautographic studies of lipid metabolism with the electron microscope have been limited by the large losses of lipid which occur during the usual dehydration and infiltration procedures. In this study different methods of processing tissue for electron microscopic radioautography have been compared with respect to the extraction of cholesterol-1, 2-3H from labeled mouse liver and nerve.In all methods primary fixation had been in 10% formalin for several months followed by washing overnight. Post-fixation for one hour in Dalton's chrome osmium and staining in 1% uranyl acetate in 10% formalin resulted in small but constant losses. Four methods were assessed: 1) graded acetone dehydration with propylene oxide and epon-araldite mixture infiltration, 2) exposure to 0.5% digitonin in 50% ethanol for 1 hour before post-fixing in osmium followed by procedure 1, 3) Durcupan embedding, and 4) limited dehydration followed by embedding in an epon:araldite mixture (modified Idelman).

Electron Microscopic Examination of a Transplantable Rat Mammary Carcinoma

1968 ◽  
Vol 26 ◽  
pp. 20-21
Author(s):  
S. Shirahama ◽  
G. C. Engle ◽  
R. M. Dutcher
Keyword(s):  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Electron Microscopic Examination ◽  
Undifferentiated Carcinoma ◽  
Uranyl Acetate ◽  
Sprague Dawley ◽  
Electron Microscopic ◽  
North West ◽  
X Irradiation ◽  
Tissue Specimens

A transplantable carcinoma was established in North West Sprague Dawley (NWSD) rats by use of X-irradiation by Engle and Spencer. The tumor was passaged through 63 generations over a period of 32 months. The original tumor, an adenocarcinoma, changed into an undifferentiated carcinoma following the 19th transplant. The tumor grew well in NWSD rats of either sex at various ages. It was invariably fatal, causing death of the host within 15 to 35 days following transplantation.Tumor, thymus, spleen, and plasma from 7 rats receiving transplants of tumor at 3 to 9 weeks of age were examined with an electron microscope at intervals of 8, 15, 22 and 30 days after transplantation. Four normal control rats of the same age were also examined. The tissues were fixed in glutaraldehyde, postfixed in osmium tetroxide and embedded in Epon. The plasma was separated from heparanized blood and processed as previously described for the tissue specimens. Sections were stained with uranyl acetate followed by lead citrate and examined with an RCA EMU-3G electron microscope.

Electron-Microscopic localization of biotinylated gastrin bound to eosinophils in the rat stomach

1994 ◽  
Vol 52 ◽  
pp. 300-301
Author(s):  
Caroline A. Miller ◽  
David H. Nichols ◽  
Richard F. Murphy
Keyword(s):  
Electron Microscope ◽  
Phosphate Buffer ◽  
Uranyl Acetate ◽  
List Type ◽  
Cerium Chloride ◽  
Rat Stomach ◽  
Electron Microscopic ◽  
Human Sequence ◽  
Acetate Lead ◽  
Microscope Slides

Gastrin is a small peptide capable of both stimulating gastric acid secretion and acting as an enteric growth factor. Known functions of eosinophils in the rat stomach are related to immunological defense. Here we demonstrate the binding of biotinylated gastrin to rat stomach eosinophils in the electron microscope. Small pieces of stomach were fixed by immersion in 4% paraformaldehyde/0.1% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 for 1 hour. The tissue was then cryoprotected in 30% sucrose/0.1 M phosphate buffer, transferred to Tissue Tek OCT compound and frozen in isopentane cooled with liquid nitrogen. Transverse cryostat sections were cut at 25 μm, thawed in PBS and free floating sections exposed to 10−5 M biotinylated 1-17 gastrin (human sequence; Peninsula Labs) for 1 hour. Controls omitted the biotinylated gastrin from this step. Sections were then rinsed 3X in PBS and exposed to either:1).a 1:50 dilution of 10 nm Extravidin colloidal gold (Sigma) for 2 hours, or2).an avidin-biotin-alkaline phosphatase complex (ABC-AP;Vector) for 1 hour. A substrate solution containing cerium chloride was used to generate an electron dense reaction product.Sections from both procedures were postfixed in 1% OsO4 in 0.1 M phosphate buffer, rinsed and dehydrated. These were then flat embedded in EMbed 812 between two microscope slides coated with Liquid Release (both from Electron Microscopy Sciences).Polymerized sections were adhered to resin blocks using super glue, cut at 70-90 nm, stained with uranyl acetate/lead citrate and observed in a Philips CM-10 electron microscope.

Electron Microscopic Study of a Spontaneous Rat Tumor

1971 ◽  
Vol 29 ◽  
pp. 322-323
Author(s):  
P. W. Cole ◽  
R. M. Jamison
Keyword(s):  
Propylene Oxide ◽  
Uranyl Acetate ◽  
Female Rat ◽  
Lead Citrate ◽  
Sprague Dawley ◽  
Electron Microscopic ◽  
Non Invasive ◽  
Ultrathin Sections ◽  
Glass Knife ◽  
Rat Tumor

A spontaneously occurring, non-invasive mammary tumor was observed in a 7-month-old, non-lactating, random-bred Sprague-Dawley female rat. The tumor was located subcutaneously in the distal mammary line as a firmly encapsulated, lobated growth. The tumor was surgically removed and immersed in Clark's buffer containing 4% glutaraldehyde. (Later conventional histochemical studies revealed this tumor to be a relatively non-differentiated fibro-adenoma.) Exterior and interior portions of the tumor were excised and fixed separately. The tissues were cut into 1 mm cubes and fixed for 4 hours in 4% glutaraldehyde. The specimens were then washed 4 times in buffer and left overnight at 4°C in the buffer. The cubes were postfixed in 2% OsO4 for 2 hours, after which they were dehydrated in a graded series of ethanol. The specimens were then washed with 2 changes of propylene oxide and perfused with a 1:1 mixture of propylene oxide-Epon 812 for 1 hour. Next, the tissue cubes were embedded in a mixture of Epon 812, DDSA, NMA, and DMP 30. Polymerization was allowed to proceed sequentially at 37°C overnight, 45°C for 8 hours, and 60°C for 24 hours. Ultrathin sections were cut with the Porter-Blum MT-2B ultramicrotome equipped with a glass knife. Sections were picked up on copper grids and stained with saturated uranyl acetate and 0.2% lead citrate. Specimens were examined in the Philips 300 electron microscope at instrumental magnifications ranging from 3,000-20,000 times.

Electron Microscopic Studies of Gerbil Testis After Vasectomy

1976 ◽  
Vol 34 ◽  
pp. 154-155
Author(s):  
S. K. MAJUMDAR ◽  
FRED KALENSCHER
Keyword(s):  
Electron Microscope ◽  
Normal Structure ◽  
Uranyl Acetate ◽  
Meriones Unguiculatus ◽  
Mongolian Gerbils ◽  
Electron Microscopic ◽  
Transmission Electron ◽  
Acetate Lead ◽  
Microscopic Studies ◽  
Ultrathin Sections

Ultrathin sections made from bilaterally vasectomized as well as bilaterally sham-operated Mongolian gerbils (Meriones unguiculatus) were examined and compared under an electron microscope in order to determine whether vasectomy has any effect upon the fine structure of the testis. The whole testes were removed and placed in Karnovsky's fixative for one hour. After this period the testes were diced into small pieces and fixed for an additional hour in the same fixative. After rinsing in distilled water and postfixed for one hour in OsO4, the tissues were embedded in Epon 812. The sections were stained with uranyl acetate-lead citrate and examined on a Philips Model 201 transmission electron microscope. Shamoperated testis exhibited normal structure of germ cells.

Electron microscopic study on the phagocytic lining cells in the cavernous body of the lamprey gill

1978 ◽  
Vol 36 (2) ◽  
pp. 448-449
Author(s):  
Kazuhito Yamaguchi ◽  
Kazuhiko Awaya
Keyword(s):  
Electron Microscope ◽  
Uranyl Acetate ◽  
Osmic Acid ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Embedded Blocks ◽  
Ultrathin Sections ◽  
Branchial Artery ◽  
Scanning Electron ◽  
Cavernous Body

The lining cells in the cavernous body, which is a branch of the afferent branchial artery, of lamprey gills were studied with transmission (TEM) and scanning electron microscope (SEM).Adult and larval lampreys, Lampertra planeri, were used in this study. Lamprey gills removed after perfusion were fixed in 2 % glutaraldehyde/formaldehyde (pH 7. 4) for 1 hour and postfixed in 1 % osmic acid. For TEM, the gills were dehydrated in ethanol and embedded in epon 812. Ultrathin sections were doubly stained with uranyl acetate and lead citrate. For SEM, most of the gills were dehydrated in ethanol and isoamylacetate and cracked in liquid nitrogen. Some of the gills dehydrated were embedded in stylene monomer. Stylene embedded blocks were cracked with a hummer and dissolved in propylene oxide. All the specimens were dried in a critical point dryer and coated 100 Å thickness with gold-paladium.The cavernous body is composed of trabeculae, between which are blood spaces, the lacunae.

Ultrastructure of Developing Granulosa and Theca Cells

1970 ◽  
Vol 28 ◽  
pp. 92-93
Author(s):  
Iracema M. Baccarini
Keyword(s):  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Ether Anesthesia ◽  
Rat Fetuses ◽  
Theca Cells ◽  
The Past ◽  
Microscopic Studies

The embryology of granulosa and theca cells is not understood thoroughly. Electron microscopic studies in the past have been concerned mainly with mature granulosa cells and less with their development.Material and Methods. Rat fetuses were removed surgically under ether anesthesia at 16-17, 17-18 and 18-19 days of gestation. Their abdominal cavities were opened, and the fetuses were placed immediately into 3% glutaraldehyde (pH 7.2) for 3 hours. During this time, the fetal ovaries were dissected under a microscope. The tissue was washed in phosphatebuffer for 24 hours, post-fixed in 1% phosphate buffered osmium tetroxide for 1-2 hours at 4°C, and embedded in Durcupan ACM (Fluka). Sections were double stained with uranyl acetate and lead citrate, and viewed in an RCA-EMU-3D electron microscope.

scholarly journals SENSITIVITY IN ELECTRON MICROSCOPE AUTORADIOGRAPHY II. EFFECT OF HEAVY METAL STAINING

1973 ◽  
Vol 21 (7) ◽  
pp. 623-627 ◽  
Author(s):  
MIRIAM M. SALPETER
Keyword(s):  
Heavy Metals ◽  
Heavy Metal ◽  
Electron Microscope ◽  
Uranyl Acetate ◽  
Protein Films ◽  
Section Thickness ◽  
Electron Microscopic ◽  
Electron Microscope Autoradiography ◽  
Electron Microscopic Autoradiography ◽  
Microscope Autoradiography

In quantitative electron microscopic autoradiography test specimens for sensitivity usually consist of plastic or protein films 500-1000 Å in thickness. For tritium, the applicability of sensitivity values derived from such specimens to biologic sections which had been stained with heavy metals was determined. It was found that, within the range of section thickness used for electron microscopic autoradiography, fixation with OsO4 followed by uranyl acetate staining increases self-absorption by less than 7% over that seen in Epon sections of the same thickness. A somewhat larger effect is seen in sections used for light microscopic autoradiography. The effect on electron microscopic autoradiographic resolution is estimated to be within 10%.

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 Block-staining tissues with potassium ferrocyanide-reduced osmium tetroxide and lead aspartate for electron microscopic radioautography.

1984 ◽  
Vol 32 (5) ◽  
pp. 552-554 ◽  
Author(s):  
B M Kopriwa
Keyword(s):  
Room Temperature ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Potassium Ferrocyanide ◽  
Electron Microscopic ◽  
En Bloc ◽  
Simple Method ◽  
Electron Microscopic Radioautography

In the hope of devising a method for prestaining tissues en bloc for electron microscopic radioautography, pieces of radioiodine-labeled liver were taken through various combinations of ferrocyanide-reduced osmium tetroxide, lead aspartate, and aqueous uranyl acetate at room temperature or at 60 degrees C. Following the tests, the method adopted for routine use was to block-stain tissues for 2 hr in potassium ferrocyanide-reduced osmium tetroxide at 4 degrees C followed by 1 hr in Walton's lead aspartate at room temperature. This simple method, which requires no manipulation before or after emulsion coating and development of the radioautographs, provides adequate contrast without inducing background fog or artifacts.

A Reproducible Selective Stain for Cardiac Sarcoplasmic Reticulum

1974 ◽  
Vol 32 ◽  
pp. 214-215
Author(s):  
R. A. Waugh ◽  
J. R. Sommer
Keyword(s):  
Complex System ◽  
Electron Microscope ◽  
Sarcoplasmic Reticulum ◽  
Transmission Electron Microscope ◽  
Electron Microscopic ◽  
Cardiac Sarcoplasmic Reticulum ◽  
Transmission Electron

Cardiac sarcoplasmic reticulum (SR) is a complex system of intracellular tubules that, due to their small size and juxtaposition to such electron-dense structures as mitochondria and myofibrils, are often inconspicuous in conventionally prepared electron microscopic material. This study reports a method with which the SR is selectively “stained” which facilitates visualizationwith the transmission electron microscope.

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