Alexafluor 488 conjugated goat anti rabbit

Overall, 1.5 × 10⁵ Vero E6 cells were seeded in a eight-well chamber (LabTek™, Nunc) and infected with WT-VSV, SARS-CoV-2, or rVSV-ΔG-spike for 24 h. Cells were then fixed with 3% paraformaldehyde (PFA) in PBS for 20 min and permeabilized with 0.5% Triton X-100 for 2 min. The fixed cells were blocked with PBS containing 2% FBS and stained with either hyperimmune rabbit serum from intravenous SARS-CoV-2 infected rabbits diluted 1:200 (in-house preparation), naive or vaccinated hamster sera diluted 1:200, or COVID-19 convalescent human sera diluted 1:200 for 1 h. After washing with PBS, cells were incubated with either Alexa Fluor 488 conjugated goat anti-rabbit at a dilution of 1:200 (Sigma, Israel, Cat# F6005), Alexa Fluor 488 conjugated goat anti-hamster at a dilution of 1:100 (Jackson, Cat# 107-545-142), or Alexa Fluor 488 conjugated goat anti-human at a dilution of 1:200 (Sigma, Cat# F0132, lot SLBR4951V). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI). Images were acquired by an Axioskop (Zeiss) equipped with a DS-iR1 camera and NIS-elements software (Nikon).

Immunofluorescence microscopy was performed on cultured cells, as previously described (31 (link)). For immunolabeling of vinculin, cultured cells were incubated with mouse anti-vinculin monoclonal antibody (1:100, V9131, Invitrogen, Waltham, MA), followed by Alexa Fluor 488-conjugated goat anti-mouse (1:100, 16-240, sigma-aldrich). The Alexa Fluor 568 Phalloidin (A12380, ThermoFisher Scientific) dissolved in 150 μL of anhydrous DMSO to yield a 400X stock was diluted by DPBS. After incubation of the secondary antibody for vimentin immunolabeling, cells were stained with diluted phalloidin stock solution for 30 min. For double-immunolabeling of myosin light chain 2 (MLC2) and phosphorylated MLC2, cultured cells were incubated with rabbit anti-MLC2 antibody (1:100, #3672, Cell signaling Technology) and mouse anti-phosphorylated MLC2 (1:100, #3675, Cell signaling Technology), followed by Alexa Fluor 488-conjugated goat anti-rabbit (1:100, 16-240, sigma-aldrich) and Alexa Fluor 568-conjugated goat anti-mouse (1:100, sigma-aldrich). Immunofluorescence microscopy was performed using a laser scanning confocal microscope (Zeiss LSM 800; Jena, Germany).