Alexa fluor 594 labeled goat anti rabbit igg

Polylysine-treated round coverslips were placed in a 24-well-plate. PMN suspension was added into the wells with a density of 2.5 × 10⁵ cells/well. Phorbol 12-myristate 13-acetate (PMA) was added to the wells. The final concentration of PMA was adjusted to 100 nmol/L. The well-plate was placed in an incubator with 5% CO₂ and incubated at 37°C for 3 h. Then, 4% paraformaldehyde was added to fix the cells at room temperature for 1 h. The cells were then washed three times with PBS-T wash buffer. Subsequently, 0.3% Triton X-100 was added to permeabilize for 15 min before blocking in 10% goat serum for 1 h. The cells were incubated in a refrigerator at 4°C overnight with PBS buffer diluted mouse-derived MPO and rabbit-derived ELA2 (Proteintech Group, Wuhan, USA). The cells were washed three times with PBS-T wash buffer and incubated in the dark for 1 h after adding Alexa Fluor® 488-labeled goat anti-mouse IgG and Alexa Fluor® 594-labeled goat anti-rabbit IgG (ZSGB-BIO, Bejing, China). The cells were again washed three times. The anti-fluorescence quenching sealing agent containing DAPI (Solarbio, Bejing, China) was added dropwise to the round coverslips, which were then removed from the well-plate and inverted on slides. The cells were visualized under a laser confocal microscope (Leica, Germany).