Fluoromount g

Immunohistochemical staining was performed as described previously with a few modifications (Lin et al., 2021 (link)). Drosophila adult testes were dissected in 1 × phosphate buffer saline (PBS) and fixed in 4% paraformaldehyde for 20 min. After washing with PBST (0.3% Triton in 1× PBS) three times, the testes were incubated in a blocking buffer (5% goat serum in 0.1% PBST) for 1 h. Next, the blocking buffer was replaced by primary antibodies mixed in PAT (1% BSA, 0.1% Triton and 0.01% sodium azide in 1× PBS) and the incubation lasted for overnight. Then, the testes were washed three times with 0.1% PBST and incubated in secondary antibodies mixed in 0.1% PBST for 1 h. After washing with 0.1% PBST three times, the tissues were stained with DAPI for 10 min and washed three times before mounting with Fluoromount-G. DAPI (#MBD0015, Sigma-Aldrich, Germany), DCP1 (#9578S, Cell signaling, USA).

Apoptosis was determined using cleaved caspase 3 IF as previously described [12 (link)]. Briefly 40,000 cells were seeded in 12 well plates on glass coverslips in triplicate. 24 hours later cells were treated with chemotherapeutic agents with or without A69 for 24 hours. After treatment, cells were fixed in 3.7% formaldehyde for 15 minutes and permeabilized in 0.3% Triton X-100 for 15 minutes. Antibodies were diluted in blocking buffer that consisted of 0.2% non-fat dry milk, 2% Bovine Serum Albumin and 0.3% Triton X-100 in phosphate buffered saline (PBS). Primary anti-cleaved caspase 3 rabbit monoclonal antibody (1:400, Cell Signaling Technology, Danvers, MA) was applied to cells for 1 hour at 37°C. Following washes in PBS, samples were incubated in goat-anti-rabbit Alexa Fluor 488 (1:1000, Life Technologies, Carlsbad, CA) and Alexa 555 conjugated Phalloidin (1:200, Life Technologies) to visualize F-actin. Slides were mounted with Fluoromount G with Hoescht (Sigma) to label cell nuclei. The percentage of positive cells was determined for each assay with at least 150 cells being counted per condition using the counting feature on an Evos Xl core microscope. Each assay was run in triplicate and repeated three times. Representative images were taken on Zeiss Axio A1 Microscope with an AxioCam MRc digital camera.

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)].