Epoxy embedding medium kit

Infected organoids were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer (1 liter of dH₂O, 21.4 g of sodium cacodylate, 1 g of MgCl₂, 0.5 g of CaCl₂, adjusted to pH 7.42 with HCl), postfixed in 1% osmium tetroxide diluted in sodium cacodylate buffer, dehydrated with an ethanol series, and then embedded with the Epoxy Embedding Medium kit (Sigma-Aldrich). After embedding, samples were cured at 65°C for 48 h. Semithin (0.5-μm) sections were cut on a Leica UCT ultramicrotome and stained with toluidine blue on a microscope slide with suitable areas selected for ultrathin 50-nm sectioning. Ultrathin sections were collected on copper grids and contrasted with uranyl acetate and lead citrate before viewing on an FEI 120-kV Spirit BioTWIN transmission electron microscope. Images were taken on an F4.15 Tietz charge-coupled device camera.

Chemicals such as Dulbecco’s Modified Eagle’s Medium (DMEM), l-Glutamine, Fetal Bovine Serum (FBS), Trypsin-EDTA solution, Antibiotic-Antimycotic solution (10000 U/mL Penicillin, 10 mg/mL Streptomycin and 25 μg/mL Amphotericin B in 0.9 % normal saline for 100 X), MTT dye, Dimethyl Sulfoxide (DMSO), Trypan blue, Dulbecco’s Phosphate Buffered Saline (DPBS), Tris-EDTA, Propidium iodide (PI), Ribonuclease A (RNase A) and Triton X-100 were purchased from Himedia, India. Molecular probes, Fluorescein isothiocyanate-Phalloidin (FITC-Phalloidin), and 4′,6- diamidino-2-phenylindole (DAPI) were obtained from ThermoFisher Scientific, USA. DCFDA, 3,3′-dihexyloxacarcocyanine iodide (DiOC₆), Rotenone, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, disodium phosphate (Na₂HPO₄) 25 % (v/v) glutaraldehyde, paraformaldehyde, osmium tetroxide, uranyl acetate, and lead citrate were purchased from Sigma Aldrich, USA. The kits used in this study such as the SOD Assay kit and Epoxy Embedding Medium Kit were procured from Sigma Aldrich, USA (Cat. No.:19106 and Cat. No.: 45359-1EA-F respectively), and the ATP Determination kit was purchased from Thermo Scientific, USA (Cat. No.: A22066). The absolute ethanol used in this study is of HPLC grade (Commercial Alcohols, Greenfield Global, Canada). All the reagents and chemicals are of analytical grade.

For the TEM examination, specimens were collected by centrifugation and treated with 2.5% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4) at 4 °C for 2 h. Subsequently, 1% osmium tetroxide was applied as a post fixative (4 °C, 1.5 h).
The sample was then dehydrated by successive dilutions of ethanol (50, 70, 90, 95 and four times 100%, each for 30 min) then dehydrated by acetone for 30 min. At the end, epoxy glue was used to implant the fixed specimens (Epoxy Embedding Medium Kit; Sigma- Aldrich, St. Louis, MO, USA). The ultramicrotome was used to cut ultra-thin and semi-thin slices (RMC PT-XL PowerTome Ultramicrotome, Wetzlar, Germany). Semi-thin (1 μm–850 nm) slices were stained with 1% toluidine blue and viewed under a light microscope using an Olympus BX61. Ultra-thin slices were cut to a thickness of 70–90 nm and stained with 2.5% uranyl acetate as the primary stain and lead citrate as the counter stain [53 ]. Finally, ultra-thin slices were examined using a JEM-1400 Plus (JEOL, Tokyo, Japan) transmission electron microscopy at the electron microscopy unit of the Faculty of Science at Alexandria University in Alexandria, Egypt.

For ultrastructural analysis of renal morphology perfusion-fixed WT and Cav1−/− kidney were subjected to additional fixation in 0,5% glutaraldehyde/PBS overnight at + 4 °C, processed for embedding using Epoxy Embedding Medium kit (Sigma-Aldrich, St. Louis, USA), and analyzed by transmission electron microscopy (Zeiss E905 or Technai^TM G2). Cellular distribution of Cav1 was analyzed by the pre-embedding technique. To this end, 30 µm thick cryostat sections from WT and Cav1−/− mice were treated with 0.5% Triton X-100 for 30 min, blocked with 5% skim milk in PBS for 30 min, and incubated with anti-Cav1 antibody for 1 h at room temperature followed by overnight incubation at + 4 °C. The corresponding HRP-conjugated secondary antibody was used for signal generation and the sections were processed for embedding in LR White resin, cut, and analyzed by transmission electron microscopy.

Mouse oviducts were fixed at room temperature in a 2% formalin/2.5% glutaraldehyde mixture buffered in 0.1 M sodium cacodylate at pH 7.4 for one hour, rinsed and fixed in 1% osmium tetroxide for another hour followed by 1% tannic acid and 1% sodium sulphite for 30 minutes respectively. Samples were then dehydrated in an ethanol series, staining en bloc with uranyl acetate at the 30% stage before embedding (Epoxy embedding medium kit – Sigma). 50 nm ultrathin sections were cut on a Leica UC6 ultramicrotome and imaged on an FEI 120 kV Spirit Biotwin TEM with a F4.15 Tietz camera.
For scanning electron microscopy, mouse oviducts were fixed as for TEM (replacing tannic acid with osmium-thiocarbohydrazide), dehydrated in an ethanol series, critical point dried in a Leica CPD300, mounted and sputter-coated with 2 nm of platinum using a Leica ACE600 evaporator. Samples were imaged on a Hitachi SU8030 SEM.

Cells seeded for 48 h in 24-multiwells (150 cells/mm²) and treated with HU-308 2.5 µM for 4 h were fixed in 0.1 M phosphate buffered 2.5% glutaraldehyde (Serva Electrophoresis, Heidelberg, Germany) and post-fixed in 1% osmium tetroxide (OsO₄) (Agar Scientific Elektron Technology, Stansted, UK) in 0.1 M phosphate buffer. After dehydration in a graded ethanol series, the samples were embedded in Epoxy Embedding Medium Kit (45349, Sigma-Aldrich, St. Gallen, Switzerland), and cut in semithin (0.5 µm) and ultra-thin (60 nm) sections, with an RMC Power-Tome ultramicrotome (Boeckeler Instruments, AZ). Semithin sections were stained in 1% Toluidine blue for 30 min and analyzed by light microscopy; ultra-thin sections were collected on 300-mesh copper grids and counterstained with 2% uranyl acetate and then Sato’s lead for TEM analysis.

Adult specimens of N. davidi were decapitated and fixed with 2.5% glutaraldehyde in a 0.1 M sodium phosphate buffer (pH 7.4, 4°C, 2h), postfixed in 2% osmium tetroxide in a 0.1 M phosphate buffer (4°C, 1.5 h) and dehydrated in a graded series of concentrations of ethanol (50, 70, 90, 95 and 4x100% each for 15 min) and acetone (15 min). Afterwards, the material was embedded in epoxy resin (Epoxy Embedding Medium Kit; Sigma). Semi- (0.8 μm thick) and ultra-thin (70 nm) sections were cut on a Leica Ultracut UCT25 ultramicrotome. Semi-thin sections were stained with 1% methylene blue in 0.5% borax and observed using an Olympus BX60 light microscope. After staining with uranyl acetate and lead citrate, ultra-thin sections were examined using a Hitachi H500 transmission electron microscope.