Validating rHcEPH Interaction via IFA

According to prior description, an immunofluorescence assay (IFA) was conducted to validate interaction [24 (link)]. In brief, 10 μg/mL rHcEPH (experimental group) or phosphate-buffered saline (PBS, control group) were used separately for a 2-h incubation of the freshly isolated PBMCs (1 × 10⁶ cells/mL) in a humidified atmosphere with 5 percent CO₂ at 37 °C. After washing, cells were allowed to rest on poly-L-lysine treated glass pieces for 15 min prior to being fixated in four percent paraformaldehyde for a quarter of hour at room temperature. Afterwards, the cells were subjected to the blockage agent (5% BSA in PBS) at 37 °C for 1 h to reduce backdrop coloring to the minimum. Cell incubation was conducted using rat anti-rHcEPH sera (1:100 dilutions; primary antibody) for 1 h. Before incubating Cy3-labeled Goat Anti-Rat IgG (1:500 dilutions; secondary antibody; Beyotime, Shanghai, China) for 45 min. The cells underwent counterstaining with DAPI (Sigma, MO, USA) for 5 min. Stained cells were imaged using a confocal laser scanning microscope (Nikon, Tokyo, Japan).