Af405

Vero cells (1.5 × 10⁴) were seeded in Nunc Lab-Tek II 8-well no. 1.5 glass chambers (Thermo Scientific) for 22 h, infected for 2 h at 37°C with ppSFV at an MOI of 10, cultured, and treated with DMSO or BBC per the standard protocol. At 8.5 h postinfection, cells were fixed with fresh 4% paraformaldehyde in phosphate-buffered saline and processed for immunostaining with or without permeabilization, as indicated in the legends, and using the following antibodies: mouse MAb E1-1 against E1 (82 (link)), anti-SFV E2/E1 rabbit PAb, mouse MAb 2-3 against Cp, mouse MAb E2-1 against E2, mouse MAb E7 against β-tubulin, and isotype and species-specific goat secondary antibodies conjugated to AF405, AF488, or AF568 (Invitrogen). Nuclei were stained with Hoechst 33342. Cells were imaged on a Leica SP8 confocal microscope with LAS X software, a 63× oil objective (numerical aperture 1.4), and HyD hybrid detectors using the same settings for DMSO and BBC samples, and image processing was performed using ImageJ v. 1.51u.

Trastuzumab and pertuzumab (Genentech) were used for staining the extracellular domains of HER2. Anti-cytokeratin 7 (Abcam, Cat# ab181598, Cambridge, UK) and goat anti-rabbit (Abcam, Cat# ab6702) were used to detect epithelial cells. Trastuzumab and pertuzumab were conjugated to Alexa Fluor 647 (AF647, Invitrogen, Cat# A20006), and goat anti-rabbit was conjugated to Alexa Fluor 405 (AF405, Invitrogen Cat# A30000) using previously published methods [97 (link)]. According to NanoDrop measurements, in all cases approximately one dye per antibody (degree of labeling ~1) was obtained for Trastuzumab and pertuzumab.