Epon araldite

Rats were perfused transcardially, under deep anesthesia, with a solution containing 2% PFA and 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (Sigma-Aldrich) pH 7.3. After fixation sciatic nerves were removed and immersed in the same fixative solution overnight at 4°C. The specimens were then postfixed in 2% OsO₄ (Sigma-Aldrich), dehydrated, and embedded in Epon-Araldite (Sigma-Aldrich). Semithin (0.5 μm) transverse sections were stained with toluidine blue and analyzed with a Axioskop200 microscope (Zeiss, Gottingen, Germany), at final 1500x magnification. To assess morphological alterations of peripheral nervous system (PNS), qualitative and quantitative analyses were performed. Usually, 5 sections for each animal nerve were analysed. At least 25 fields for each section, corresponding to 25% of the total nerve area, were acquired [50 (link)]. The number of myelinated fibers was counted, normalized per field (in μm²), and expressed as mean ± SEM of data.

To qualitatively analyze cellulose crystallinity under polarized light, internode segments were first cut on a vibratome into 100 μm sections, fixed in 2% glutaraldehyde and then embedded in Epon/Araldite (Sigma-Aldrich, St. Louis, MO, United States) before slicing into 0.5 μm sections on an Reichert-Jung, Ultracut E microtome (Vienna, Austria). Polarized light microscopy takes advantage of variation in the passage of light through crystalline structures to uncover discrete differences in crystallinity configurations. Specifically, the LC-PolScope (CRI, Cambridge, MA, United States) employed for this assay uses polarized light to measure birefringent retardance and the intensity of the images generated directly correlates to the retardance value, thus qualitative differences in crystallinity can be observed. Several stem samples from each HIF were imaged and analyzed.

CD4+ naïve T cells co-cultured with CD70-NC and CD70-KO C666 cells in the lipid-depleted or normal co-culture culture system after 3 days were washed, gently centrifuged, and re-suspended in PBS, then cells were fixed in 2.5% glutaraldehyde (Sigma-Aldrich), postfixed in 2% osmium tetroxide (Sigma-Aldrich), stained with uranyl acetate (Sigma-Aldrich), and dehydrated with a graded ethanol series. Sections were cut after embedding in Epon Araldite (Sigma-Aldrich), collected on uncoated nickel grids after sectioning and the mitochondria were observed and photographed using Philips CM-100 Transmission Electron Microscope. The sample preparation and electron microscopy scanning were performed by the Electron Microscope Unit at the University of Hong Kong.

For ultrastructural analysis, HUVE and HCM cells were treated with 1 mM 5-FU for 72 or 96 hours. Growth media alone and containing DMSO were used as controls. Cells were fixed in a mixture of 2% paraformaldehyde and 2% glutaraldehyde, post-fixed in 1% osmium tetroxide, and embedded in Epon-Araldite (Sigma-Aldrich). After counterstaining with uranyl acetate and lead citrate, thin sections were examined with a Morgagni electron microscope (Philips, Eindhowen, NL) at 7100X magnification.

DAB stained cell suspensions were post-fixed with 1% aqueous OsO4 for 1 hour on ice, subsequently rinsed three times with pure water and dehydrated in a sequence of ethanol solutions (70% up to 100%), followed by incubation in 100% propylene oxide and embedding in Epon/Araldite (Sigma-Aldrich, Buchs, Switzerland). Samples were polymerized at 60°C for 24h. Thin sections were imaged without post-staining as well as after post-staining with aqueous uranyl acetate (2%) and Reynolds lead citrate.

For TEM observations, five females were paralyzed by exposure to cold temperatures (−18 °C) for 60 s, then immediately immersed into a solution of glutaraldehyde and paraformaldehyde 2.5% in 0.1 M cacodylate buffer +5% sucrose, pH 7.2–7.3. Each antenna was detached from its base, single antennomeres were isolated and reduced in size by cutting them in two parts to facilitate fixative penetration and then left at 4 °C for 2 h. The specimens were kept at 4 °C overnight in the same buffer, then they were post-fixed in 1% OsO4 (osmium tetroxide) for 1 h at 4 °C and rinsed in the same buffer. Dehydration, in a graded ethanol series from 60% to 99%, was followed by embedding in Epon-Araldite (Sigma-Aldrich, Dorset, UK) with propylene oxide as a bridging solvent. Thin sections were taken with a diamond knife on a LKB “Nova” ultramicrotome (LKB, Stockholm, Sweden) and mounted on 50 formvar-coated mesh grids. Then, sections on the grids were stained with uranyl acetate (20 min, room temperature) and lead citrate (5 min, room temperature). Finally, the sections were observed with a Philips^® EM 208 (Thermo Fisher Scientific Inc., Hillsboro, OR, USA). Digital pictures (1376 × 1032 pixels, 8 bit, uncompressed greyscale TIFF files) were obtained using a high-resolution digital camera MegaViewIII (SIS^®) (SIS, Muenster, Germany ) connected to the TEM.