4.6. Transmission Electron Microscopy

The fungal mycelium was transferred to Eppendorf tubes containing 1 mL of fixative (a mixture of 4% formaldehyde and 2% glutaraldehyde in 0.1 M cacodylic buffer). The specimens were fixed overnight at 4 °C and washed three times in 0.1 M cacodylic buffer followed by 1 h incubation in a 1% OsO₄ (0223B, Polysciences Inc., Warrington, PA, USA) water solution. After contrast, the samples were dehydrated using increasing ethanol concentrations and transferred to propylene oxide (00236-1, Polysciences, Inc., USA). After 20 min, the samples were infiltrated overnight with a propylene oxide:resin (Epon Resin 828, 02334, Polysciences, Inc., USA) mixture (1:1) and incubated for 3 h in 100% resin. Subsequently, the resin was exchanged, and the samples were incubated overnight at 37 °C. The sections (60 nm) were cut using an ultramicrotome (Leica EM UC7, Austria), and the images were collected using a JEOL JEM2100 HT transmission electron microscope (JEOL, Tokyo Japan).