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Antibodies to Glutathione: Production, Characterization, and Immunocytochemical Application to the Central Nervous System
Published in Christopher A. Shaw, Glutathione in the Nervous System, 2018
Ole P. Hjelle, E. Rinvik, D. Huster, W. Reichelt, Ole P. Ottersen
It has been demonstrated in the present review and in previous studies (Hjelle et al. 1994; Usami et al. 1996) that fixed glutathione can be recognized by the antibodies even after embedding in an acrylic (Lowicryl) or epoxy (Durcupan) resin. This has important implications as it permits the application of postembedding immunogold procedures. Besides providing a high anatomical resolution, the latter procedures afford possibilities for quantitative analyses, based on particle counting (Usami et al. 1996; Huster et al. 1997). The particle counts can be regarded as reliable indicators of the glutathione concentration, because the postembedding labeling is restricted to the section surface and thus unaffected by penetration barriers in the tissue (Ottersen 1989a). The development of electron-microscopic calibration systems similar to those designed for other small molecules (Ottersen 1989b; Nagelhus, Lehmann, and Ottersen 1993) should allow the particle counts to be translated into millimolar concentrations of the peptide. Thus the postembedding procedure offers the potential of estimating the concentration of glutathione in subcellular compartments, at an anatomical resolution in the low nanometer range.
Mitochondrial organization and structure are compromised in fibroblasts from patients with Huntington’s disease
Published in Ultrastructural Pathology, 2022
Marie Vanisova, Hana Stufkova, Michaela Kohoutova, Tereza Rakosnikova, Jana Krizova, Jiri Klempir, Irena Rysankova, Jan Roth, Jiri Zeman, Hana Hansikova
Fibroblasts were cultivated as mentioned previously to 90% confluence and fixed in phosphate-buffered saline (PBS) containing 2% (w/V) potassium permanganate for 15 min at room temperature (RT). After fixation, cells were dehydrated in an ethanol series (for 10 min in 50%, 70%, and 90% ethanol, for 30 min in 100% ethanol) at RT. Dehydrated cells were incubated in propylenoxide (2x15 min) and embedded in Durcupan Epon (Electron Microscopy Sciences, Hatfield, PA) (Durcupan Epon:Propyleonxide 1:1 for 2 hours, Durcupan Epon:Propylenoxide 1:3 overnight at 60°C). Polymerized blocks were sectioned by Ultracut III ultramicrotome (Reichert, Depew, NY) and 600–900 Å thick slices were stained with lead citrate and uranyl acetate.4 Pictures were taken on a transmission electron microscope JEOL JEM-1200 EX.
C. elegans MAGU-2/Mpp5 homolog regulates epidermal phagocytosis and synapse density
Published in Journal of Neurogenetics, 2020
Salvatore J. Cherra, Alexandr Goncharov, Daniela Boassa, Mark Ellisman, Yishi Jin
To investigate the cellular interactions between the epidermis and the nerve cord, we used miniSOG to label cholinergic neurons using tissue-specific promoters. miniSOG-labeled cellular membranes were visualized by EM after fixation and photo-oxidation. Adult animals were placed in 2% glutaraldehyde, 2% paraformaldehyde solution in 100 mM sodium cacodylate buffer. After the animals stopped moving, they were cut in half and incubated for 1 h on ice. The samples were washed in 100 mM sodium cacodylate buffer and then blocked in 50 mM glycine, 10 mM potassium cyanide, 20 mM aminotriazole in 100 mM sodium cacodylate buffer for 2 h on ice. The samples were placed in a MatTek culture dish containing ice-cold 2.5 mM oxygentated diaminobenzadine and 10 mM HCl in 100 mM sodium cacodylate buffer and illuminated with blue light using a Leica SPEII confocal microscope for 20 min. The samples were then washed in 100 mM sodium cacodylate buffer and post fixed overnight at 4 °C in 2% osmium tetroxide in 100 mM sodium cacodylate buffer. The following day samples were rinsed with ice-cold ddH2O, dehydrated with ethanol and acetone, embedded in Durcupan, and baked for 3 days at 60 °C.
The effects of nano-sized PbO on biomarkers of membrane disruption and DNA damage in a sub-chronic inhalation study on mice
Published in Nanotoxicology, 2020
Lucie Bláhová, Zuzana Nováková, Zbyněk Večeřa, Lucie Vrlíková, Bohumil Dočekal, Jana Dumková, Kamil Křůmal, Pavel Mikuška, Marcela Buchtová, Aleš Hampl, Klára Hilscherová, Luděk Bláha
Samples of lungs were fixed in 3% glutaraldehyde for 24 h, washed three times in a 0.1 M cacodylate buffer and post-fixed in a 1% OsO4 solution for 1.5 h. After washing in a cacodylate buffer, all samples were dehydrated in a series of increasing concentrations of ethanol, followed by acetone and then embedded in epoxy resin Durcupan. For EM analysis, 60-nanometer thick sections were prepared from selected parts of samples. Sections were cut using an ultramicrotome Leica EM UC6 (Leica Mikrosysteme GmbH, Vienna, Austria) and placed on formvar-coated nickel grids. Some sections were used without further contrasting for analysis of nanoparticles in TEM. Other sections were contrasted with uranyl citrate and lead acetate for studying cell architecture. All sections were observed using a Morgagni™ 268 TEM (FEI Company, Eindhoven, Netherlands), designated structures were measured using the software iTEM. Photographs were taken using a Veleta CCD camera (Olympus, Münster, Germany).