Zo 1 primary antibody

After treatment with the toxins and porcine colostrum, the cells were washed in PBS, fixed in 1% methanol-free formaldehyde for 30 min, permeabilised using 0.1% Triton X-100 for 10 min and blocked in 5% foetal calf serum for 30 min. The F-actin rhodamine phalloidin probe and zonula occludens 1 (ZO-1) rat monoclonal antibody were applied on the coverslips containing IPEC-J2 cells and incubated for two hours at room temperature. The cells were then washed in PBS and stained with goat anti-rat IgG Alexa Fluor 594 to detect the primary antibody against ZO-1. The nuclear DNA was stained with DAPI. Fluorescence signals from F-actin and ZO-1 were visualised using a fluorescent confocal microscope (Zeiss LSM 710, Jena, Germany) equipped with a 20x dry objective lens, NA 0.8 and allowing spectral resolution of up to five stainings in parallel, in the range 405 nm to 633 nm. The ZO-1 primary antibody was purchased from Santa Cruz Biotechnology (Heidelberg, Germany). The F-actin probe and goat anti-rat secondary antibodies, DAPI and methanol-free formaldehyde were purchased from ThermoFisher Scientific, Invitrogen™ (Darmstadt, Germany).

An immunofluorescence procedure was performed as previously described [51 (link)].
Nasal tissue sections were incubated with ZO-1 primary antibody (Santa Cruz Biotechnology, Dallas, TX, USA; #sc-33725). Slices were examined and images were acquired at 40× magnification using a Leica DM2000 microscope (Leica, Milan, Italy).