Propylene oxide embed 812

Wild-type, ΔrlmA, and complementing strains were gown in liquid YG medium during 24 hr. Cells were processed essentially as described previously (Bom et al. 2015 (link)) with modifications. Briefly, the mycelia were fixed in 0.1 M sodium phosphate buffer (pH 7.4) containing 2.5% (v/v) of glutaraldehyde for 24 hr at 4°. Samples were encapsulated in agar (2% w/v) and subjected to fixation (1% OsO₄), contrasting (1% uranyl acetate), ethanol dehydration, and a two-step infiltration process with propylene oxide/EMbed 812 (Electron Microscopy Sciences) of 16 and 3 hr at room temperature. Additional infiltration was provided under vacuum at room temperature before embedding in BEEM capsules (Electron Microscopy Sciences) and polymerizing at 60° for 72 hr. Semithin (0.5 µm) survey sections were stained with toluidine blue to identify the areas of best cell density. Ultrathin sections (60 nm) were prepared and stained with uranyl acetate (1%) and lead citrate (2%). Transmission electron microscopy (TEM) images were obtained using a Philips CM-120 electron microscope at an acceleration voltage of 120 kV using a MegaView3 camera and iTEM 5.0 software (Olympus Soft Imaging Solutions GmbH). Cell wall thicknesses of 50 sections of different germlings were measured using magnification of 66,000 × and iTEM 5.0 software analysis.

Wild-type and ΔmsbA strains (1 × 10⁷ conidia) were grown in liquid MM for 24 h at 37°C prior to exposure to CFW (100 μg/ml) for 2 h. Cells were processed essentially as described previously with modifications (35 (link)). Briefly, mycelium was fixed in 0.1 M sodium phosphate buffer (pH 7.4)-2.5% (vol/vol) glutaraldehyde for 24 h at 4°C. Samples were encapsulated in 2% (wt/vol) agar and subjected to fixation (1% OsO₄), contrast (1% uranyl acetate), ethanol dehydration, and a two-step infiltration process with propylene oxide-EMbed 812 (Electron Microscopy Sciences) of 16 h and 3 h at room temperature. Additional infiltration was provided under vacuum at room temperature before embedding in BEEM capsules (Electron Microscopy Sciences) and polymerization at 60°C for 72 h. Semithin (0.5-mm) survey sections were stained with toluidine blue to identify the areas of greatest cell density. Ultrathin sections (60 nm) were prepared and stained with uranyl acetate (1%) and lead citrate (2%). Transmission electron microscopy (TEM) images were obtained using a Tecnai G2-12-SpiritBiotwin FEI electron microscope at an acceleration voltage of 120 kV (Center of Microscopy from UFMG, Brazil) using a charge-coupled device (CCD) camera. Cell wall thicknesses of 50 sections of different germlings were measured using magnification of ×26,500 and ImageJ software analysis (13 (link)).