Alexa fluor 488 conjugated anti mouse and 594 conjugated anti rabbit secondary antibodies

Slides were placed in 24-well plates and then covered with Matrigel (BD). Bulk cells, GSCs, or DGCs were seeded at a density of 1 × 10⁵ cells per well. After 24 h, the cells were infected with ZIKV-LAV at a multiplicity of infection (MOI) of 0.1 for 1 h, and the culture medium was then refreshed. At the indicated times, the supernatants were collected for virus titration by RT-qPCR as described above. The bulk cells and GSCs were incubated with the same primary and secondary antibodies used for the immunofluorescence analysis of the brain cryosections. The DGCs were incubated with the mouse anti-ZIKV E protein (Abcam) (1:500 dilution) and rabbit anti-GFAP (Dako) (1:500 dilution) primary antibodies overnight at 4°C and then with Alexa Fluor 488-conjugated anti-mouse and 594-conjugated anti-rabbit secondary antibodies (Thermo Fisher) (1:300 dilution) for 2 h at 37°C. The cells were subsequently incubated with DAPI (1:1,000 dilution) for 10 min. Images were acquired by confocal laser scanning microscopy (Zeiss).

Apoptosis in tumor tissue was detected by cleaved caspase 3 staining and a DeadEnd colorimetric TUNEL system kit (Promega). For cleaved caspase 3 staining, the brain cryosections were incubated with the primary mouse anti-ZIKV E protein antibody or rabbit anti-cleaved caspase 3 antibody (CST) (1:200 dilution) and Alexa Fluor 488-conjugated anti-mouse and 594-conjugated anti-rabbit secondary antibodies (Thermo Fisher) (1:300 dilution). Finally, the sections were incubated with DAPI (1:1,000 dilution) for 10 min. A DeadEnd colorimetric TUNEL system kit (Promega) was utilized according to the manufacturer’s instructions. Images were acquired by confocal laser scanning microscopy (Zeiss).