Small animal optical imaging system in vivo xtreme 2

BV2 cells inoculated in confocal dishes were treated with HSV-1 and NAMO for indicated time, the cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 5% bovine serum albumin. Subsequently, the cells were incubated with p65 antibody overnight at 4°C and were treated with Alexa Fluor 488 (green)-labeled secondary antibody (A32790, Thermo Fisher Scientific) for 1 h at room temperature. The nucleus were stained with DAPI (C1006, Beyotime, China). Finally, fluorescent images were obtained by a confocal laser scan microscope (LSM 510 meta, Zeiss). For mitophagy assay, BV2 cells were transfected with GFP-LC3B (2 μg) or mitoKeima (1 μg) plasmids using Lipofectamine 3000 transfection reagent (ThermoFisher, L3000015) for 24 h before infected with HSV-1 and treated with NAMO for another 12 h. The cells transfected with GFP-LC3B plasmids were fixed and stained with Mito-Tracker Red (ThermoFisher, M7512) for 0.5 h. The cells transfected with mitoKeima plasmids were untreated. Finally, fluorescent images were obtained by a confocal laser scan microscope (LSM 510 meta, Zeiss). For in vivo imaging, normal or ABX-treated mice were infected with EGFP-HSV-1, and the in vivo EGFP distribution image was taken with the Bruker Small Animal Optical Imaging System (In‐Vivo Xtreme II; Billerica, MA).