Goat anti rabbit red alexa 594

Infected Vero cells on glass coverslips were fixed with ice-cold methanol for 5 min, at room temperature. After fixation, cells were washed three times with PBS and then blocked and permeabilized for at least 1 h with a blocking buffer (PBS, 0.1% saponin, 1% BSA). For double immunostaining, the samples were incubated in blocking solution for 1 hour, at room temperature, with 1:1000 dilution of primary rabbit anti W. chondrophila antibodies and 1:200 mouse antibody dilutions targeting the protein of interest. After three washes with PBS, coverslips were incubated for 1 hour in blocking solution containing 1:1000 dilutions of secondary antibodies, Goat anti-mouse green Alexa 488 and Goat anti-rabbit red Alexa 594 (Thermo Fischer, Waltham, MA, USA), and 150 ng/mL DAPI (Molecular Probes). Coverslips were washed three times with PBS, once with water, and were mounted onto glass slides, using Mowiol (Sigma-Aldrich).

Polyclonal mouse antibodies against W. chondrophila were produced by our group, as previously described [16 (link)]. Secondary antibodies, Goat anti-mouse green Alexa 488, and Goat anti-rabbit red Alexa 594 were purchased from Thermo Fischer Scientific (Waltham, MA, USA). DAPI was obtained from Molecular Probes (Grand Island, NY). The antibiotics penicillin and phosphomycin, and 2, 2-Bipyridyl were purchased from Sigma-Aldrich (St Louis, MO, USA).