Vectashield mounting medium

HCE cells (3 × 10⁵ cells/well) were cultured on sterile cover glass in a 6-well plate with KGM at 37 °C with 5% CO₂ for 24 h. The next day, A. castellanii trophozoites (5 × 10⁵ cells/well) or cysts (5 × 10⁵ cells/well) were added and co-cultured for 4 h. F. solani, P. aeruginosa, and S. aureus were cultured in the early exponential phase (OD_600nm = 0.8) and co-cultured with HCE cells and A. castellanii for 1 h. Cells were washed with ice-cold phosphate-buffered saline (PBS) and then fixed with 100% methanol (chilled at −20 °C) for 5 min. The cells were washed three times with PBS and permeabilized with PBST (0.2% Triton X-100 in PBS) for 5 min at room temperature (RT). Cells were blocked for 30 min at RT in blocking buffer (1% bovine serum albumin and 22.52 mg/mL glycine in PBST). Cells were incubated overnight at 4 °C with the ACAP or PBP antibodies in blocking buffer (1:200) and probed with CruzFluorTM 555 (CFL 555)-conjugated anti-rabbit IgG (1:400) (Santa Cruz, Dallas, TX, USA) for 2 h at RT. After washing three times with PBS, cells were stained with VECTASHIELD mounting medium (Abcam, Burlingame, CA, USA) and observed under a fluorescence microscope (Leica DMi8, Wetzlar, Germany).

HCE cells (3×10⁵ cells) were cultured on sterile cover glass in a 6-well plate. The following day, they were co-cultured with A. castellanii trophozoites (5×10⁵ cells), and cysts (5×10⁵ cells) for 5 h at 37°C with 5% CO₂. F. solani, P. aeruginosa, and S. aureus were cultured in liquid broths until early exponential phase (OD_600nm = 0.8), which were subsequently co-cultured with HCE cells and A. castellanii for 1 h. After washing with PBS, the cells were fixed with ice-cold 100% methanol for 5 min at RT. Cells were repeatedly washed and permeabilized with PBST (1 X PBS containing either 0.1% Tween 20) for 10 min at RT. Afterward, cells were washed and subsequently blocked using blocking buffer (1% bovine serum albumin and 22.52 mg/ml glycine in PBST) for 30 min at RT. Cells were incubated overnight at 4°C with CE antibody (1:200) in blocking buffer and probed with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IgG (1:400) for 1 h at RT. After repeated washing, cells were stained with VECTASHIELD mounting medium (Abcam, Burlingame, CA, USA) and observed under a fluorescent microscope (Leica DMi8, Wetzlar, Germany).