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Ultrastructural Immunocytochemistry
Published in Joan Gil, Models of Lung Disease, 2020
Samuel S. Spicer, Bradley A. Schulte
This approach has important advantages. Penetration of staining reagents into a fixed ultrathin cryosection undoubtedly exceeds that into a resin of sufficient hardness for ultramicrotomy. In addition, the frozen sections are spared the adverse action on the antigenicity that results from exposure to catalyzing chemicals and high temperature during polymerization of resin. The cryo method still suffers from the denaturing action of chemical fixatives on the antigen, and only freeze-drying avoids this problem.
Composition of The Chromaffin Cell
Published in Stephen W. Carmichael, Susan L. Stoddard, The Adrenal Medulla 1986 - 1988, 2017
Stephen W. Carmichael, Susan L. Stoddard
Instrumentation and techniques have been developed by Ornberg (1985) for the cryo-ultramicrotomy of isolated bovine adrenal chromaffin cells. Cells were frozen with the liquid helium-cooled copper block method. X-ray microanalysis and electron energy loss spectroscopy were used to measure both light and heavy element contents of the chromaffin vesicle. Values for elements such as phosphorus and nitrogen were in reasonable agreement with the known biochemical composition of chromaffin vesicles.
Neuroviral Infections
Published in Sunit K. Singh, Daniel Růžek, Neuroviral Infections, 2013
In conventional electron microscopic technique the volumes of tissue samples are small. Therefore, in experimental neuroanatomy and neuropathology the introduction of perfusion fixation of the CNS with osmium tetroxide (Palay et al. 1962), later, with glutaraldehyde and paraformaldehyde, meant a great step forward. A further development of this technique was the embedding of entire brain or head slices of mice and rats in synthetic resin, cutting the whole surface area into thin sections by “hot knife microtomy,” selection of appropriate regions by light microcopy and excision of the selected small regions from a subsequent 200-300 µm thick tissue slice, and processing them to ultramicrotomy (McGee-Russell et al. 1990).
The effect of pulsed radiofrequency application on nerve healing after sciatic nerve anastomosis in rats
Published in Ultrastructural Pathology, 2022
Uğur Ö. Bayır, Recep Aksu, Özlem Öz Gergin, Gozde Ozge Onder, Leman Sencar, Eray Günay, Arzu H. Yay, İbrahim Karaman, Cihangir Bicer, Sait Polat
Nerve tissue pieces taken for electron microscopic evaluation were fixed in a 5% glutaraldehyde solution prepared with Millonig’s phosphate buffer for 4 hours. Tissues were then washed twice in Millonig’s phosphate buffer for 10 minutes and fixated for a second time with 1% osmium tetraoxide (OsO4) solution. After two hours of fixation with OsO4, the tissues were washed twice with phosphate buffer for 10 minutes. Then, they were dehydrated with an increasing series of ethyl alcohol. The tissues were put in a propylene oxide solution two times for 15 minutes for transparency, and the tissue pieces were kept in the mixture consisting of 1 volume of propylene oxide and 1 volume of resin for two times for 30 minutes each after the transparency processes were completed. Tissue pieces were placed into resin after these processes and rotated in the rotator for a night. Tissue pieces were embedded in Beem capsules using resin and kept in a 60°C incubator for 48 hours. Thin sections of 50 nm thickness were taken from the blocks with Ultracut S ultramicrotomy. Sections were stained with uranyl acetate and lead citrate solutions. Stained sections were examined by Transmission Electron Microscope (JEOL-JEM 1400, Tokyo, Japan), and micrographs were obtained.
Leptin receptor defect with diabetes causes skeletal muscle atrophy in female obese Zucker rats where peculiar depots networked with mitochondrial damages
Published in Ultrastructural Pathology, 2021
Jacques Gilloteaux, Charles Nicaise, Lindsay Sprimont, John Bissler, Judith A Finkelstein, Warren R Payne
The rat’s corpses used for LM were rapidly perfused with 3.5% buffered glutaraldehyde solution (0.1 M Na cacodylate, pH 7.35, at room temperature for 15 min), as in71 and the 3 contralateral legs, sectioned with tibialis muscles still in situ, were excised to undergo the same fixation that continued for 2 h at 4°C. Washed in buffered sucrose solution, segments of muscle specimens were thinned into muscle fiber bundles, postfixed in 1.5% aqueous osmium tetroxide solution and processed for transmission (TEM) electron microscopy after embedment in PolyBed epoxy resin (Polysciences, Warrington PA.). One-µm thick sections, stained by toluidine blue, were observed with an Olympus BX51 light photomicroscope (Olympus America, Melville NY) to select areas for ultramicrotomy. Ultrathin sections were collected on 50, 75- and 100-mesh hexagonal copper grids (SPI, West Chester PA), contrasted by uranyl acetate and lead citrate prior to be examined in a JEOL 100 S electron microscope (JEOL USA, Inc, Peabody, MA).
Isolation and characterization of a novel thermophile; Bacillus haynesii, applied for the green synthesis of ZnO nanoparticles
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Suriya Rehman, B. Rabindran Jermy, Sultan Akhtar, J. Francis Borgio, Sayed Abdul Azeez, Vijaya Ravinayagam, Reem Al Jindan, Zainab Hassan Alsalem, Abdullah Buhameid, Adil Gani
Cell morphology and structural details of the isolate were investigated by SEM and TEM. The TEM was regulated at accelerating voltage of 80 kV. A 24 h culture was used for the morphology and structural details of CDL3, while 48 h culture was used for spore assessment. For TEM, cell suspension and spore preparation were analyzed using chemical fixation [27]. Briefly, the suspension of cell/spores was centrifuged to obtain specimen pellets. The pellets were then fixed in the primary fixative (a mixture of 4% glutaraldehyde and 2.5% paraformaldehyde buffered with 0.1 M PIPES). The supernatant was then removed by centrifuge and rinsed 2–3 times in PIPES buffer. The pellets were cut into 1–2 mm cubes. The cubes were further processed with a second fixative (buffered osmium tetra-oxide and incubated for 1 h at room temperature. The osmium tetroxide was removed and rinsed with distilled water. Later, samples were dehydrated by graded ethanol (30, 50, 70 and 90%) each for 10 min. The specimens were passed through absolute ethanol three times, each for 10 min. The fixed cells/spores were then exposed to propylene oxide and infiltered with propylene oxide. Cells/spores were embedded in pure resin mixture in beam capsule and cured in an oven at 60–70 °C for 48 h. Afterwards, ultrathin sections of each specimen were made by ultramicrotomy and few sections were transferred onto TEM grids. Finally, TEM grids holding cell/spore sections were stained by contrast enhancing agent uranyl acetate [28,29]. Stained grids were mounted into TEM and taken several micrographs of the spores/cells at different magnifications.