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 SENSITIVITY IN ELECTRON MICROSCOPE AUTORADIOGRAPHY FOR 125I

1974 ◽  
Vol 22 (2) ◽  
pp. 80-87 ◽  
Author(s):  
HELEN C. FERTUCK ◽  
MIRIAM M. SALPETER
Keyword(s):  
Heavy Metal ◽  
Electron Microscope ◽  
Radiation Dose ◽  
Dose Dependence ◽  
Specimen Thickness ◽  
Section Thickness ◽  
Electron Microscope Autoradiography ◽  
Absorption Studies ◽  
Microscope Autoradiography ◽  
Better Than

Sensitivity in electron microscope autoradiography was determined for 125I. Values are given using Ilford L4 and Kodak NTE emulsions combined with different developers. The extent of self-absorption as a function of section thickness and heavy metal staining and the effect of radiation dose (dose dependence) were assessed. It was found that the over-all efficiency for 125I was better than that for tritium and that, as is the case with tritium, there is a distinct "dose dependence" especially when Microdol X is the developer. Self-absorption studies indicate that self-absorption is increased by about 15% when the specimen thickness is increased from 300 to 1000 Å. An increase of under 15% is also introduced by heavy metal staining of sections in this thickness range.

scholarly journals Resolution in electron microscope autoradiography. III. Iodine-125, the effect of heavy metal staining, and a reassessment of critical parameters.

1977 ◽  
Vol 72 (1) ◽  
pp. 161-173 ◽  
Author(s):  
M M Salpeter ◽  
H C Fertuck ◽  
E E Salpeter
Keyword(s):  
Heavy Metal ◽  
Electron Microscope ◽  
Silver Halide ◽  
Uranyl Acetate ◽  
Limiting Factors ◽  
Critical Parameters ◽  
Limiting Factor ◽  
Electron Microscope Autoradiography ◽  
Iodine 125 ◽  
General Aspects

Resolution for 125I-labeled specimens under electron microscope (EM) autoradiographic conditions was assessed experimentally. With this isotope the size of the silver halide crystal was the most important resolution-limiting factor. Heavy metal staining such as is routinely used in preparing animal tissues for EM autoradiography produced an improvement in resolution of approximately 15-20%. For a 500-1,000-A biological tissue section fixed with OsO4 and stained with uranyl acetate, we obtained resolution (half distance, HD) values of approximately 800 +/- 120 A using Ilford L4 emulsion and 500 +/- 70 A using a Kodak NTE-type emulsion. General aspects of resolution-limiting factors and comparison with 3H and 14C values are discussed.

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.

Incorporation of Cholesterol into Peripheral Nerve Myelin

1972 ◽  
Vol 30 ◽  
pp. 334-335
Author(s):  
Frank A. Rawlins
Keyword(s):  
Electron Microscope ◽  
Sciatic Nerve ◽  
Electron Microscope Autoradiography ◽  
Myelin Sheaths ◽  
Microscope Autoradiography ◽  
Grain Distribution ◽  
Sciatic Nerves ◽  
Autoradiographic Analysis ◽  
Old Mice ◽  
Short Times

Several speculations exist as to the site of incorporation of preformed molecules into myelin. The possibility that an autoradiographic analysis of cholesterol-1,2-H3 incorporation at very short times after injection might shed some light in the solution of that problem led to the present experiment.Cholesterol-1,2-H3 was injected intraperitoneally into 24 tenday old mice. The animals were then sacrificed at 10,20,30,40,60,90,120 and 180 min after the injection and the sciatic nerves were processed for electron microscope autoradiography. To analyze the grain distribution in the autoradiograms of cross and longitudinal sections from each sciatic nerve myelin sheaths were subdivided into three compartments named: outer 1/3, middle 1/3 and inner 1/3 compartments.It was found that twenty min. after the injection of cholesterol -1.2-H3 (Figs. 1 and 2), 55% of the total number of grains (t.n.g) found in myelin were within the outer 1/3 compartment, 9% were within the middle 1/3 and 36% within the inner 1/3 compartment

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.

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