Epon 812 epoxy resin

The samples for ultrastructural studies were fixed in a 3.6% buffered glutaraldehyde solution at pH 7.4 (Poly Scientific R&D Corporation, Bay Shore, NY1176, USA). They were then embedded in an Epon 812 epoxy resin (Sigma Aldrich, Poznan, Poland) and sliced into 1 µm-thick slices using an ultramicrotome. Subsequently, they were stained with toluidine blue and examined using a light microscope. Such ultra-thin sections were examined using electron microscopy. The photographic documentation was collected using an Opton 900 transmission electron microscope (Zeiss, Oberkochen, Germany).
The diagram of the above study flow description is shown in Figure 1.

Control SD rats or SD rats at 4 days after sciatic nerve crush were perfused with a fixative containing 1% paraformaldehyde and 1.25% glutaraldehyde. Sciatic nerve tissue specimens were harvested and fixed with 4% glutaraldehyde, post-fixed with 1% osmium tetroxide, washed, dehydrated, embedded in Epon 812 epoxy resin (Sigma-Aldrich, St. Louis, MO, United States), and cut into ultra-thin sections. Sciatic nerve sections were stained with lead citrate and uranyl acetate. Distal nerve segments were observed under a transmission electron microscope (JEOL Ltd., Tokyo, Japan) at magnifications of × 0.7 k, × 1.0 k, and × 30.0 k.

Cells were fixed in PBS with 2.5% glutaraldehyde/paraformaldehyde for 45 min. The cells were washed and post-fixed with 1% osmium tetroxide (OsO4). Then, the cells were dehydrated with an ascending series of ethanol and embedded in Epon 812 epoxy resin (Sigma, St. Louis, MO, USA). Ultrathin (~70 nm) sections were cut with an ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany), collected on copper grids, and stained with uranyl acetate and lead citrate. Samples were observed on a JEOL 10/10 transmission electron microscope (JEOL, Tokyo, Japan).

HCT-8 and SK-N-AS cells (5 × 10⁵) were pelleted by centrifugation at 600× g and washed twice in isotonic PBS. The pellets were fixed in Karnovsky’s fixative (cacodylate-buffered 4% glutaraldehyde and 2% paraformaldehyde (pH 7.4) and post-fixed with a solution of 1% OsO₄ (Electron Microscopy Sciences, Hatfield, PA, USA). The samples were embedded in Epon 812 epoxy-resin (Sigma-Aldrich SRL, Milan, Italy) and cut with an RMC MT-X ultramicrotome (EMME3, Milan, Italy), collecting ultrathin sections (~700 nm thick) that were afterward stained with UranyLess (Electron Microscopy Sciences) and alkaline bismuth subnitrate solutions. The observation was performed with a JEM-1010 electron microscope (Jeol, Tokyo, Japan) at 80 Kv, and photomicrographs were acquired at 25,000× magnification using a MegaView III high-resolution digital camera equipped with an image analysis software Radius 2.2 (Emsis, Muenster, Germany). Morphometric analysis was expressed as the ratio between vesicles and total cell area (n = 5) and quantified using ImageJ software (https://fiji.sc (accessed on 5 August 2020)).

Cotyledons from mature B. napus seeds were isolated and stained according to the method of Siloto et al. (2006 (link)). The central part of the semicircular cotyledons was fixed with 2.5% glutaraldehyde in a 0.1 M phosphate buffer (pH 6.8) for 24 h, and postfixed with a 1% osmium tetroxide solution for an additional 2 h. After dehydration by using the acetone series (20, 50, 70, 90, and 3 × 100% v/v), the cotyledons were filtrated and subsequently embedded in 100% (w/v) EPON-812 epoxy resin (Sigma-Aldrich, America). Ultrathin sections (0.7 μm) were prepared by using a diamond knife on a UC6 Ultratome (Leica, Germany) onto copper grids, and stained with uranyl acetate and lead citrate. Images were acquired by using an H-7650 transmission electron microscope (TEM) (Hitachi, Japan).