Epon 812 resin

For the TEM studies, a morphogenic callus of Brachypodium was fixed in 2.5% glutaraldehyde in a 0.1 M sodium phosphate buffer (PB; pH 7.4) at 4 °C for 24 h. After washing in phosphate buffered saline (PBS, 5 times, 30 min each), the material was postfixed in 1% osmium tetroxide in a 0.1 M PB (4 °C, 24 h), rinsed with the same buffer and dehydrated in a graded concentration series of ethanol (30%, 50%, 70%, 90%, 96%, and 100%, each for 15 min) and acetone (two times, 15 min each) followed by infiltration in mixtures of acetone and Epon 812 resin (3:1, 1:1, and 1:3) (Polysciences, Eppelheim, Germany). Then, the material was embedded in Epon 812 resin and polymerized into resin blocks at 60 °C for 48 h. Ultra-thin (70 nm) sections were cut with a diamond knife on an Ultracut UCT25 (Leica, Wetzlar, Germany) ultramicrotome. After contrast staining with uranyl acetate and lead citrate, the sections were examined using a H500 transmission electron microscope (Hitachi, Tokyo, Japan).

The assessment of nerve pathology followed our established protocol (Chiang et al., 2005 (link)). The sural nerves were collected at the level of the trifurcation of the sciatic nerve and then fixed in 5% glutaraldehyde in 0.1 M phosphate buffer at 4°C overnight. The tissues were post-fixed in 2% osmic acid for 2 h at room temperature, dehydrated, and embedded in Epon 812 resin (Polysciences, Philadelphia, PA, United States). Ultrathin (70 nm) sections were cut on a Reichert Ultracut E (Leica, Wetzlar, Germany), stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (H7100, Hitachi, Tokyo, Japan).

Sural nerves were fixed with 5% glutaraldehyde in 0.1 M PB for 2 h and then embedded with Epon 812 resin (Polyscience, Philadelphia, PA, USA). Sural nerve pathology was assessed in accordance with our previously established protocol [43 (link)]. Semithin sections (0.5 μm) were cut with an ultramicrotome (Reichert Ultracut E, Leica, Wetzlar, Germany) and stained with toluidine blue. For morphometric analyses, the sections were photographed at 400× magnification under an ECLIPSE Ci-S microscope (Nikon, Tokyo, Japan). Image-Pro Plus software (Media Cybernetics) was used to calculate axonal density (axons/mm²), mean axonal diameter (μm), and axonal area distribution, as previously described [43 (link)].

To analyze the membrane structure of a phagosome containing one single yeast, macrophages were stimulated with H. capsulatum at a low yeast-to-macrophage ratio (MOI = 2) for 30 min. After stimulation, cells were washed by Dulbecco's Phosphate-Buffered Saline (DPBS) and detached by using Accutase Cell Detachment Solution (BD Biosciences). Cells for TEM were processed as described previously (37 (link)). In brief, cells were pelleted, fixed in 5% glutaraldehyde, post-fixed with 2% osmic acid, dehydrated, and embedded in Epon 812 resin (Polysciences). Ultrathin sections (70 nm) were cut and stained with uranyl acetate and lead citrate before observation under a transmission electron microscope (H7100, Hitachi).

The processing of nerve pathology followed our established protocol49 (link). The sciatic nerves at the gluteal level were collected. Tissues were fixed in 5% glutaraldehyde in 0.1 M PB overnight, postfixed in 2% osmic acid for 2 h at room temperature, dehydrated with a graded series of alcohol, and embedded in Epon 812 resin (Polyscience, Philadelphia, PA). Cross-sections of 1 μm were cut on an ultramicrotome (Leica, Wetzlar, Germany), dried on slides using a hot plate, stained with toluidine blue, and observed under a light microscope.

We used DRG tissues to investigate the pathology of ER stress. Briefly, we dissected lumbar DRG tissues and postfixed the tissues in 2% osmium tetroxide for 2 h, dehydrated them through a graded ethanol series, and embedded them in Epon 812 resin (Polyscience, Philadelphia, PA, USA). Thin sections (50 nm) were stained with uranyl acetate and lead citrate, after which we observed them using an electron microscope (Hitachi, Tokyo, Japan) and photographed them.