Alexa fluor 555 conjugated goat anti rabbit igg secondary antibody

The Vero cells were washed with PBS, dislodged with 0.25% Trypsin–EDTA (Sigma life sciences), and seeded into 96‐well imaging plates (Greiner) at a density of 10⁴ cells per well in culture media (Dulbecco's modified Eagle medium (DMEM) containing 10% FBS, 1% penicillin and streptomycin, 1% l‐glutamine and 1% non‐essential amino acids). Cells were incubated for 24 h to allow time for adherence. Virus or cells were treated with polymeric solutions, diluted in media, 1 h prior to infections. Cells were subsequently infected with SARS‐CoV‐2 virus England 2 stock 10⁶ IUml⁻¹ (kind gift from Christine Bruce, Public Health England) diluted 1/150 in culture media. Cells were fixed in ice‐cold MeOH after infection. Cells were then washed in PBS and stained with rabbit anti‐SARS‐CoV‐2 spike protein, subunit 1 (The Native Antigen Company), followed by Alexa Fluor 555‐conjugated goat anti‐rabbit IgG secondary antibody (Invitrogen, Thermofisher). Cell nuclei were visualized with Hoechst 33342 (Thermofisher). Cells were washed with PBS and then imaged and analyzed using a ThermoScientific CellInsight CX5 High‐Content Screening (HCS) platform. Infected cells were scored by perinuclear fluorescence above a set threshold determined by positive (untreated) and negative (uninfected) controls.

Briefly, 2 × 10⁵ cells were seeded in µ-slide 8-well chambered coverslips (Ibidi, Gräfelfing, Germany) and incubated in culturing medium containing either DMSO or mebendazole for 24 h. In addition, 5 µm cryosections of tumor samples of the animal studies that have been air-dried for 10 min were used. Cells or tissue sections were fixed in 4% paraformaldehyde/PBS for 15 min, permeabilized with 0.5% Triton X-100 for 1 h and then blocked in PBST containing 3% BSA and 0.1% glycine for 10 min. Slides were then incubated overnight at 4 °C with mouse anti-α-tubulin (clone DM1A) (Sigma-Aldrich), Ki67 (Abcam, Cambridge, UK) and cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA) antibodies in 5% BSA/PBS at dilutions of 1:100, 1:250, and 1:100, respectively. The next day, following serial washing steps, slides were incubated with Alexa Fluor 555-conjugated goat anti-rabbit IgG secondary antibody (Invitrogen) in a dilution of 1:250 in 5% BSA and 0.1% Triton X-100. Slides were counterstained with 1 µg/mL Hoechst 33342 (Invitrogen) at room temperature in the dark for 1 h. Images of tubulin staining were captured with the 40× objective of the Axiovert 200M microscope equipped with an AxioCam MRm (Zeiss, Jena, Germany) and abnormal spindles were counted by ImageJ. Ki67 and caspase-3 stainings were captured with the EVOS M7000 microscope (Invitrogen).