Confocal Microscopy of Antifungal Effects

For confocal microscopy, a 1 × 10⁶ cells/mL fungal suspension was treated with the same concentrations of nonyl, fluconazole, and amphotericin B in 24-well plates (Kasvi) containing sterile round coverslips. After 96 h of incubation, the plates were centrifuged, the culture medium was gently removed, and the wells were washed with PBS after successive centrifugation steps. A working solution of Calcofluor White (Sigma-Aldrich) was prepared at a concentration of 100 mg/L, and 30 µL of this solution was added to the wells. The plates were then reincubated at 37 °C for 45 min and protected from light. The wells were then washed with sterile PBS, and the plates were centrifuged. Coverslips were carefully removed and poured into 4 μL of Fluoromount-G (Sigma-Aldrich), previously deposited on microscope slides for observation under a confocal microscope, the ZEISS LSM 800 with Airyscan with image capture and processing software ZEN BLUE 2.3 System [20 (link),43 (link)].