Jem 1200em

Three-week-old nodules were fixed and processed using the low-temperature embedding procedure as previously described (Tsyganova et al., 2009 (link)). For light microscopy, 0.5-μm-thick, resin-embedded sections were cut with a glass knife and collected on slides. Specimens were stained in 5% Toluidine blue in 0.1 mM sodium borate. Sections were examined on a Nikon Eclipse 800 with a Nikon Coolpix 995 digital camera (Nikon Corp., Tokyo, Japan). For transmission electron microscopy, 90–100-nm-thick ultrathin sections were collected on copper grids with 4% pyroxylin and carbon. The grids with sections were counterstained in 2% aqueous uranyl acetate for 1 h followed by lead citrate for 1 min. The sections of nodules were examined and photographed in a JEM-1200 EM (JEOL Corp., Tokyo, Japan) transmission electron microscope at 80 kV.

The samples were fixed with 2% paraformaldehyde (PFA), 2% glutaraldehyde (Distilled EM grade, Electron Microscopy Sciences, Hatfield, PA) in 0.1 M PBS pH 7.4 at 4°C overnight. After the fixation, the samples were rinsed three times with 0.1 M PBS for 30 minutes, followed by post-fixation with 2% osmium tetroxide(OsO₄) in PBS at 4°C for two hours. The samples were then infiltrated with propylene oxide (PO) twice for 15 min and put them into a mixture of PO and resin (Quetol-812; Nisshin EM Co.,Tokyo, Japan) for one hour, followed by keeping the cap of tube open, and PO was volatilized overnight. The samples was transferred to a fresh 100% resin, and polymerized at 60°C for 48 hours. The blocks were ultra-thin sectioned at 70 nm with a diamond knife using a ultramicrotome (ULTRACUT UCT; Leica, Wetzlar, Germany) and sections were placed on copper grids. They were stained with 2% uranyl acetate at room temparature for 15 min. and then rinsed with distilled water followed by being secondary-stained with Lead stain solution (Sigma-Aldrich) at room temparature for three minutes. The grids were observed by a transmission electron microscope (JEM-1200EM; JEOL Ltd., Akishima, Japan) at an acceleration voltage of 80 kv. Digital images (2048×2048pixels) were taken with a CCD camera (VELETA; Olympus Soft Imaging Solutions GmbH, Münster, Germany).