Alexa fluor 488 labeled goat anti mouse 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).

BHK-21 cells electroporated with corresponding vRNA were seeded onto cover slips in 24-well plate-format and cultured at 37°C in 5% CO₂. At the indicated time points, the cover slips were washed once with PBS (phosphate buffered saline, pH 7.4) and fixed with acetone/methanol (v/v: 3/7) at 4°C. The cover slips were incubated with the anti-dengue envelope protein monoclonal antibody 2A10G6 at 37°C for 1 hr for the cells transfected with DENV4 RNA, and anti-ZIKV envelope protein mAb clone 0302156 (1:1000 diluted, BioFront Technologies, Tallahassee, Florida, USA) was used for the cells transfected with ZIKV RNA. After the incubation with primary antibodies, the cover slips were washed with PBS for three times. AlexaFluor 488-labeled goat anti-mouse IgG (1:200 diluted, zsbio, Beijing, China) was then added, and after a 1-h incubation, the cover slips were washed as described above. For cell nuclei staining, 4', 6-diamidino-2-phenylindole (DAPI, 0.5 ng/μl) was added onto the cover slips and incubated for 5 min. An Olympus BX51 microscope under the control of DP72 software was utilized for image capture.