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Immunocytochemical Detection Systems
Published in Lars-Inge Larsson, Immunocytochemistry: Theory and Practice, 2020
Uranyl acetate is commonly used to produce constrast in electron microscopy. Sternberger et al.326 have employed uranium-labeled antibodies for immunocytochemistry (immunouranium method). To obtain better visualization of the labeled antibodies, osmium was bridged to them by thiocarbohydrazide.326 An application of the immunouranium method to Rh antibodies was published by Toma et al.344
Carbohydrate Histochemistry
Published in Joan Gil, Models of Lung Disease, 2020
Bradley A. Schulte, Russell A. Harley, Samuel S. Spicer
Ultrastructural postembedment methods for demonstrating complex carbohydrates in situ can be grouped into three categories based on the nature of the reactive moiety stained. A first category is composed of techniques aimed at detecting glycosubstances by the reactivity of hydroxyl groups or of vicinal diols present in sugars. All of these methods, references to which can be found in a recent review (Thomopoulos et al., 1987), use as a first step a mineral acid, most commonly and preferentially periodic acid, which acts to oxidize the vicinal diol groups or hydroxyl groups of sugars. The resulting dialdehyde or aldehyde group is then exposed to any one of a number of aldehyde reactive agents, which, if electron opaque, can be visualized directly in the electron microscope or complexed with an electron-opaque heavy metal. The Thiery technique (1967) used extensively in our laboratory involves a periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) sequence and extends the light microscopic PAS procedure to the electron microscopic level, achieving greatly increased sensitivity. This method deposits electron-opaque silver deposits at the site of periodate-engendered aldehydes. The greatest sensitivity with the PA-TCH-SP method has been observed in tissues fixed with glutaraldehyde and embedded in nonepoxy resins such as a styrene-methacrylate resin mixture (Thomopoulos et al., 1983a) and LR White (unpublished observations).
Morphology changes in the cochlea of impulse noise-induced hidden hearing loss
Published in Acta Oto-Laryngologica, 2022
Guowei Qi, Lei Shi, Handai Qin, Qingqing Jiang, Weiwei Guo, Ning Yu, Dongyi Han, Shiming Yang
After the electrophysiological measurement, the guinea pig was sacrificed, and its cochlea was used for transmission electron microscopy observation. The cochlea was perfused with 2.5% (vol/vol) glutaraldehyde and immersed in the fixative for 12 h at 4 °C. Then decalcification in 10% EDTA for 48 h. The basilar membrane was carefully dissected and fixed by 2.5% glutaraldehyde with Phosphate Buffer (PB) (0.1 M, pH 7.4), washed four times in PB. Then basilar membranes were first immersed in 1% (wt/vol) OsO4 and 1.5% (wt/vol) potassium ferricyanide aqueous solution at 4 °C for one h. After washing, they were incubated in filtered 1% thiocarbohydrazide (TCH) aqueous solutions (Sigma-Aldrich) at room temperature for 30 min, 1% unbuffered OsO4 aqueous solution at 4 °C for one h and 1% UA aqueous solution at 4 °C overnight. Then tissues were dehydrated through graded alcohol (30, 50, 70, 80, 90, 100%, 100%, 10 min each) into pure acetone (2 × 10min). Samples were infiltrated in graded mixtures (3:1, 1:1, 1:3) of acetone and SPI-PON812 resin (19.6 mL SPI-PON812, 6.6 mL DDSA, and 13.8 mL NMA), then changed pure resin. Finally, cells were embedded in pure resin with 1.5% BDMA and polymerized for 12 h at 45 °C, 48 h at 60 °C. The ultrathin sections (70 nm thick) were sectioned with a microtome (Leica EM UC6), stained by lead citrate, and examined by a transmission electron microscope (FEI Tecnai Spirit120kV).
Canalicular system reorganization during mouse platelet activation as revealed by 3D ultrastructural analysis
Published in Platelets, 2021
Irina D. Pokrovskaya, Michael Tobin, Rohan Desai, Smita Joshi, Jeffrey A. Kamykowski, Guofeng Zhang, Maria A. Aronova, Sidney W. Whiteheart, Richard D. Leapman, Brian Storrie
Isolated platelet suspensions were further fixed using 0.1 M cacodylate buffer containing 2.5% glutaraldehyde and 2 mM CaCl2 for 1 h in ice. Cells were washed three times with cold 0.1 M sodium cacodylate buffer containing 2 mM CaCl2 and spun at 600 × g for 5 min. Samples were fixed in 3% potassium ferrocyanide in 0.3 M cacodylate buffer with 4 mM CaCl2 combined with an equal volume of 4% osmium tetroxide for 1 h in ice. After washing five times with H2O samples were then placed in a 0.22 μm-Millipore-filtered 1% thiocarbohydrazide (TCH) solution in ddH2O for 20 min following five washes with double-distilled water (ddH2O) at RT each for 3 min. Samples were fixed in 2% osmium tetroxide in ddH2O for 30 min at RT following five washes with ddH2O at RT each for 3 min and then placed in 1% uranyl acetate (aqueous) for overnight at 4°C. The next day, samples were washed five times with ddH2O at RT each for 3 min and processed for en bloc Walton’s lead aspartate staining at 60°C for 30 min following five washes with ddH2O at RT each for 3 min. Samples were dehydrated and proceed for resin embedding.
Histological study of costal cartilage after transplantation and reasons for avoidance of postoperative resorption and retention of cartilage structure in rats
Published in Journal of Plastic Surgery and Hand Surgery, 2018
Yukiko Rikimaru-Nishi, Hideaki Rikimaru, Shinichiro Hashiguchi, Tomonoshin Kanazawa, Keisuke Ohta, Kei-Ichiro Nakamura, Kensuke Kiyokawa
Specimens were cut into small cubes and fixed with half Karnovsky fixative (2.5% glutaraldehyde and 2% paraformaldehyde solution) in 0.1 M PB (pH 7.4) followed by post-fixation with ferrocyanate and 1% OsO4. The specimens were then treated with 1% thiocarbohydrazide and immersed in a 1% OsO4 solution, followed by further staining with Walton’s lead aspartate solution. They were then dehydrated in a graded ethanol series, infiltrated with epoxy resin (EPON812, TAAB, Berkshire, UK) and polymerized at 60 °C for 72 h [8,15]. The resin blocks were trimmed down to a 2 × 4-mm area. The surface of each embedded specimen was exposed by trimming the resin block with a diamond knife and the specimen was mounted in a special holder and set on the stage of the FIB/SEM apparatus. Serial images of the block face were acquired by repeated cycles of sample surface milling using a focused gallium ion beam (milling step: 100 nm, 1000 cycles) and by acquisition of compositional contrast SEM images from back scattered electrons (landing energy, 2.5 kV). Images were reconstructed that covered the area from the normal perichondrium to the internal region of the cartilage for control specimens in the CWP group, and the area from the perichondrium-like tissues to the internal region of the cartilage for 8-week specimens in the CWOP group. A total of 3600 block face images were obtained per specimen.