scholarly journals A METHOD FOR INTRACELLULAR AUTORADIOGRAPHY IN THE ELECTRON MICROSCOPE

1961 ◽  
Vol 10 (4) ◽  
pp. 577-587 ◽  
Author(s):  
M. H. Silk ◽  
A. O. Hawtrey ◽  
I. M. Spence ◽  
J. H. S. Gear
Keyword(s):  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Red Light ◽  
Kidney Cells ◽  
Sodium Thiosulphate ◽  
Aqueous Sodium ◽  
Fine Wire ◽  
Ultrathin Sections ◽  
Bromine Vapor ◽  
Silver Metal

A technic is described for high resolution intracellular autoradiography in the electron microscope. Cultures of LLC-MK2 monkey kidney cells were incubated for 72 hours in a medium containing 0.4 µcurie per ml of thymidine-H3. After labeling, the cells were fixed with osmium tetroxide and embedded in methacrylate. Ultrathin sections of the labeled tissue were taken up on Formvar-coated and carbon-stabilized electron microscope grids. A 150 to 450 A layer of silver metal was then evaporated onto the tissue. The coated grids were exposed to bromine vapor for 1.5 to 2 minutes under red light, allowed to dry for 1 minute, and then covered with a thin film of 1 per cent aqueous gelatin applied by means of a fine wire loop lowered over the grid supported on a glass peg. For autoradiographic exposure, the grids were stored 50 days in a light-proof container at 4°C with calcium chloride desiccant. Development was carried out for 5 minutes at 20°C in Promicrol (May and Baker, England) diluted 1:1 with water, followed by a 1 minute water wash and fixation for 2.5 minutes in 15 per cent aqueous sodium thiosulphate. After removal of the gelatin by immersion for 16 hours in water at 37°C, the autoradiograms were dried and examined in the electron microscope. Ultrastructural detail was fairly well defined and the cytoplasm of each labeled cell was covered with an electron opaque deposit of silver, suggesting that a polynucleotide containing thymidine may be synthesized in the cytoplasm. The matter is discussed.

scholarly journals The Membranes of the Basal Labyrinth in Kidney Cells of the Stickleback, Gasterosteus Aculeatus, Studied in Ultrathin Sections and Freeze-Etch Replicas

1974 ◽  
Vol 14 (3) ◽  
pp. 587-609
Author(s):  
S. E. WENDELAARBONGA ◽  
M. VEENHUIS
Keyword(s):  
Membrane Structure ◽  
Osmium Tetroxide ◽  
Cell Membranes ◽  
Gasterosteus Aculeatus ◽  
Outer Cell ◽  
Kidney Cells ◽  
Thin Sections ◽  
Protein Model ◽  
Basal Labyrinth ◽  
Ultrathin Sections

The structure of the basal labyrinth in kidney cells of freshwater sticklebacks was studied in ultrathin sections (after fixation with permanganate, osmium tetroxide, and combinations of glutaraldehyde with osmium tetroxide) and in freeze-etch replicas (after pretreatment with glutaraldehyde and/or glycerol, or without pretreatment). The structure of the basal labyrinth in sticklebacks, and probably in other teleost species, differs essentially from the type of labyrinth found in kidney cells of mammals like the rat. In the latter animals, the space enclosed by the membranes of the labyrinth is intercellular. In the stickleback the labyrinth consists of an intracellular system of branched membranes lining narrow saccular spaces. These spaces communicate with the exterior of the cells by means of small pores, located in the lateral and basal parts of the outer cell membranes. All chemical fixation procedures used introduced specific structural artifacts. It is concluded that the structure of the basal labyrinth is relatively well preserved after fixation with potassium permanganate, with a mixture of glutaraldehyde and osmium tetroxide, or with osmium tetroxide when applied for 10 min only. The unit-membrane structure was, however, absent after all procedures involving osmium tetroxide. In freeze-etch replicas determinations were made of the numbers of small particles covering the surfaces and fracture faces of the membranes of the basal labyrinth and of the outer cell membranes. The numbers per unit area of surface proved to be markedly constant and specific for each of the four faces of both types of membranes. Specific differences were found between the particle densities of the outer cell membranes and the membranes of the basal labyrinth. This finding points to functional differences between these types of membranes. Particle densities were not influenced by pre-incubation with glycerol. After fixation with glutaraldehyde, the particles adhering to the outer and inner surfaces had decreased in number. It is concluded from this study that membrane structure, as revealed in thin sections as well as in freeze-etch replicas, is consistent with Singer's ‘fluid lipid-crystal protein’ model.

An attempt to get an EEL filter image of biological sections using a spectrometer and a scanning device

1990 ◽  
Vol 48 (2) ◽  
pp. 22-23
Author(s):  
Yutaka Futaesaku ◽  
Kachiko Sekiya ◽  
Michiko Ono ◽  
Atsushi Nakamura ◽  
Yoshiko Nakamura ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Cultured Cells ◽  
Scanning Transmission Electron Microscope ◽  
Light Elements ◽  
Combined System ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Ultrathin Sections ◽  
Electron Energy Loss

An amount of light elements contained in biological specimens is barely a few, so that the detection of them in the ultrathin sections is imposible. An analysis of electron energy loss (EEL) filter image is an ideal to detect such as phosphorus including in the sections. CEM-902 is a commercially available electron microscope to use for a such purpose. The authers attempted to get EEL filter images using the combined system of a conventional scanning transmission electron microscope (STEM) with EELS analyzer.The cultured cells with calcified substances and the liver, kidney, thyroid gland were used for biological specimens. There were fixed with 2.5% glutaraldehyde and with or without 1% osmium tetroxide, and then embedded in epoxy resin. 40 run thick sections were analyzed without any staining and carbon coating using a Carl Zeiss CEM-902 or a JEOL 2000EX with a EELS analyzer and a Tracor-Northern TN-5500, and with a Gatan cryo-transfer holder.

Ultrastructure of Angiosarcoma of Mouse Tail

1980 ◽  
Vol 38 ◽  
pp. 740-741
Author(s):  
White Yvonne ◽  
Winslow Sheldon ◽  
James W. Townsend ◽  
Neil A. Littlefield
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Light Microscopy ◽  
Toluidine Blue ◽  
Female Mouse ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Lead Citrate ◽  
Ultrathin Sections ◽  
Resin Mixture

Spontaneous neoplasms rarely occur on the tails of BALB/cStCrlfC3H/Nctr mice, but the neoplasm most frequently observed is a locally invasive non-metastatic angiosarcoma. In this case a female mouse weighing 33.2 g, 706 days of age, presented a soft, red, irregularly-shaped mass, measuring 18 mm in its greatest dimension, in the subcutis of the base of the tail. A portion of the tail tumor was taken for electron microscopy and the remainder was processed for light microscopy. The tissue processed for electron microscopy was fixed in 4% cacodyl ate-buffered glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol solutions, and embedded in Epon-Araldite resin mixture. Sections of 1 μm were stained with toluidine blue for light microscopy and ultrathin sections of 100 nm were stained with uranyl acetate and lead citrate, then examined with a Philips EM201 electron microscope .

X-ray microanalytical and enzyme-cytochemical study on vesical malacoplakia

1978 ◽  
Vol 36 (2) ◽  
pp. 646-647
Author(s):  
Masami Hokano ◽  
Tsunao Oh-I ◽  
Yoshie Narita ◽  
Hiroshi Sassa ◽  
Saburo Suzuki
Keyword(s):  
Electron Microscopy ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Granulomatous Inflammation ◽  
Uranyl Acetate ◽  
The Other ◽  
Lead Citrate ◽  
Cytochemical Study ◽  
X Ray ◽  
Ultrathin Sections

Malacoplakia is a kind of granulomatous inflammation and characterized by the presence of calcium-stain positive granules (Michaelis-Gutmann bodies, hereinafter abbreviated as M-G bodies ) in the macrophages.In this report we want to say about the following articles:the ultrastruc tural findings in four cases of vesical malacoplakia;the ultrastructural morphogenesis of the M-G bodies;X-ray microanalytical studies which examine the change of the chemical component of M-G bodies, according to their developing stages; andacid phosphatase activity of lysosome within the malacoplakic macrophages.Biopsy materials taken from four cases with vesical malacoplakia were divided into 2 parts; one for a light microscopy and the other for an electron microscopy. The specimen for an electron microscopy was fixed in glutaraldehyde and osmium tetroxide, dehydrated in ethanol and then embedded in Epon 812. Ultrathin sections were doublestained with lead citrate and uranyl acetate and then subjected to routin TEM observation. For X-ray microanalysis was mounted an energy dispersive X-ray microanalyzer on a JEM-100 C type electron microscope. Ultrathin sections for microanalysis were cut 100-200nm thich and not stained.

Electron microscopy of the changes in the sperm head curvature of the palm squirrel (Funambulus Penanti)

1993 ◽  
Vol 51 ◽  
pp. 334-335
Author(s):  
S. R. Bawa ◽  
R. Bawa ◽  
H. K. Bains
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Uranyl Acetate ◽  
Sperm Head ◽  
Lead Citrate ◽  
Transmission Electron ◽  
Ultrathin Sections ◽  
Epididymal Spermatozoa

Examination of ultrathin sections of the spermatozoa recovered from the epididymis of the Indian palm squirrel (Funambulus penanti) indicates that the sperm head undergoes changes in its curvature during epididymal transit.Testis and epididymis of an adult male squirrel were dissected and small pieces of tissue fixed in 2.0% glutaraldehyde in 0.1 M phosphate buffer and post-fixed in osmium tetroxide. After dehydration in graded acetone the material was embedded in Araldite. Ultrathin sections were cut on a Reichert Jung Ultracut, picked-up on copper grids, stained with Reynold’s lead citrate-uranyl acetate and examined with a JEOL 1200 EX transmission electron microscope.Ultrathin sections of the caput epididymal spermatozoa reveal that their plasma membrane is adherent to the underlying acrosome (Figure 1). When these spermatozoa reach the corpus epididymis the plasma membrane surrounding the head becomes ruffled (Figure 2). The lifting-up of the plasma membrane around the head is restricted to the posterior bend of the acrosome.

Effects of gossypol acetic acid, a male antifertility drug, on the epididymis of DBA/2J mice

1989 ◽  
Vol 47 ◽  
pp. 1086-1087
Author(s):  
S. K. Majumdar ◽  
D. L. Wicks
Keyword(s):  
Acetic Acid ◽  
Electron Microscope ◽  
Osmium Tetroxide ◽  
Reproductive Organs ◽  
Human Beings ◽  
Male Contraceptive ◽  
Cotton Plants ◽  
Gossypol Acetic Acid ◽  
Ultrathin Sections ◽  
Sigma Chemical

Recent studies have demonstrated that gossypol acetic acid, a phenolic compound isolated from cotton plants, is a promising reversible male contraceptive in rats and in human beings. Since very little information on the light and ultrastructural effects of the compound on the male reproductive organs in mice is available, this study was undertaken to investigate the effects of the agent on epididymal tissue and spermatozoa in DBA/2J mice.Gossypol acetic acid was purchased from Sigma Chemical Co., MO. The agent, mixed in olive oil, was administered orally to four groups of mice. Each group received either 0 (control) , 10,20 or 40 mg/kg of the agent twice per week for periods up to 96 days. Animals were sacrificed in pairs at 3,6,9 and 12 weeks. Motility, viability and sperm numbers were determined. Epididymis was fixed in 2% glutaraldehyde, post fixed in 1% osmium tetroxide, dehydrated in an acetone series and embedded in Epon 812. Ultrathin sections were stained and examined under the Philips 200 electron microscope.

Further Observations on the Virus of Epizootic Diarrhea of Infant Mice. An Electron Microscopic Study

1967 ◽  
Vol 25 ◽  
pp. 110-111
Author(s):  
W. G. Banfield ◽  
G. Kasnic ◽  
J. H. Blackwell
Keyword(s):  
Electron Microscope ◽  
Infected Cell ◽  
Microscopic Study ◽  
Intestinal Epithelium ◽  
Ultrastructural Study ◽  
Osmium Tetroxide ◽  
Lead Citrate ◽  
Electron Microscopic ◽  
Virus Particles ◽  
Infected Cells

An ultrastructural study of the intestinal epithelium of mice infected with the agent of epizootic diarrhea of infant mice (EDIM virus) was first performed by Adams and Kraft. We have extended their observations and have found developmental forms of the virus and associated structures not reported by them.Three-day-old NLM strain mice were infected with EDIM virus and killed 48 to 168 hours later. Specimens of bowel were fixed in glutaraldehyde, post fixed in osmium tetroxide and embedded in epon. Sections were stained with uranyl magnesium acetate followed by lead citrate and examined in an updated RCA EMU-3F electron microscope.The cells containing virus particles (infected) are at the tips of the villi and occur throughout the intestine from duodenum through colon. All developmental forms of the virus are present from 48 to 168 hours after infection. Figure 1 is of cells without virus particles and figure 2 is of an infected cell. The nucleus and cytoplasm of the infected cells appear clearer than the cells without virus particles.

Ultrastructural comparison of prolactin adenomas of pituitary in men and women

1988 ◽  
Vol 46 ◽  
pp. 346-347
Author(s):  
R.C. Caughey ◽  
U.P. Kalyan-Raman
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Phosphate Buffer ◽  
Pituitary Adenomas ◽  
Osmium Tetroxide ◽  
Secretory Granules ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Men And Women ◽  
Transmission Electron

Prolactin producing pituitary adenomas are ultrastructurally characterized by secretory granules varying in size (150-300nm), abundance of endoplasmic reticulum, and misplaced exocytosis. They are also subclassified as sparsely or densely granulated according to the amount of granules present. The hormone levels in men and women vary, being higher in men; so also the symptoms vary between both sexes. In order to understand this variation, we studied 21 prolactin producing pituitary adenomas by transmission electron microscope. This was out of a total of 80 pituitary adenomas. There were 6 men and 15 women in this group of 21 prolactinomas.All of the pituitary adenomas were fixed in 2.5% glutaraldehyde, rinsed in Millonig's phosphate buffer, and post fixed with 1% osmium tetroxide. They were then en bloc stained with 0.5% uranyl acetate, rinsed with Walpole's non-phosphate buffer, dehydrated with graded series of ethanols and embedded with Epon 812 epoxy resin.

Ultrastructure of Testicular Spermatozoon of the Fish Oreochromis Niloticus

1988 ◽  
Vol 46 ◽  
pp. 278-279
Author(s):  
T. Guha ◽  
A. Q. Siddiqui ◽  
P. F. Prentis
Keyword(s):  
Electron Microscope ◽  
Oreochromis Niloticus ◽  
Osmium Tetroxide ◽  
Sperm Cells ◽  
Adult Fish ◽  
Testicular Sperm ◽  
Thin Sections ◽  
Important Fish ◽  
Testicular Spermatozoon ◽  
Diamond Knife

Tilapia, Oreochromis niloticus, is an economically important fish in Saudi Arabia. Elucidation of reproductive biology of this species is necessary for successful breeding program. In this paper we describe fine structure of testicular sperm cells in O, niloticus.Testes from young adult fish were fixed in gluteraldehyde (2%) and osmium tetroxide (1%), both in cacodyl ate buffer. Specimens were processed in the conventional way for electron microscopy and thin sections of tissues (obtained by cutting the blocks with a diamond knife) were stained by ura- nyl acetate and lead citrate. These were examined in a Carl Zeiss electron microscope operated at 40 kV to 60 kV. Sperm cells were obtained from testes by squeezing them in cacodyl ate buffer. They were fixed in gluteraldehyde (2%) in the same buffer, air dried, gold coated and then examined in a Philips scanning electron microscope (SEM) operated at 25kV.The spermatozoon of O. niloticus is consisting of head, midpiece and tail (Fig. 1).

Selective Staining of Acid Mucopolysaccharides By Ruthenium Red

1968 ◽  
Vol 26 ◽  
pp. 38-39
Author(s):  
J. H. Luft
Keyword(s):  
Electron Microscope ◽  
Light Microscopy ◽  
Light Microscope ◽  
Osmium Tetroxide ◽  
Single Cells ◽  
Ruthenium Red ◽  
Collagen Fibers ◽  
Selective Staining ◽  
Pectic Substances ◽  
Solid Tissues

Ruthenium red is one of the few completely inorganic dyes used to stain tissues for light microscopy. This novelty is enhanced by ignorance regarding its staining mechanism. However, its continued usefulness in botany for demonstrating pectic substances attests to selectivity of some sort. Whether understood or not, histochemists continue to be grateful for small favors.Ruthenium red can also be used with the electron microscope. If single cells are exposed to ruthenium red solution, sufficient mass can be bound to produce observable density in the electron microscope. Generally, this effect is not useful with solid tissues because the contrast is wasted on the damaged cells at the block surface, with little dye diffusing more than 25-50 μ into the interior. Although these traces of ruthenium red which penetrate between and around cells are visible in the light microscope, they produce negligible contrast in the electron microscope. However, its presence can be amplified by a reaction with osmium tetroxide, probably catalytically, to be easily visible by EM. Now the density is clearly seen to be extracellular and closely associated with collagen fibers (Fig. 1).

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