Alexa fluor 594 goat anti mouse igm secondary antibody

Cells were grown on coverslips and after oxidative stress/recovery treatments they were washed twice in ice‐cold PBS. Next, the cells were fixed in 10% PBS‐buffered formaldehyde for 30 min. at room temperature, subsequently washed with PBS. To avoid formaldehyde autofluorescence, the coverslips were quenched with 50 mM ammonium chloride for 10 min. Cells were permeabilized with 0.1% Triton‐X 100 for 5 min. Non‐specific sites were blocked with 5% bovine serum albumin (Sigma‐Aldrich) in PBS for 5 min. and then the coverslips were incubated at room temperature with CTD110.6 monoclonal antibody for 30 min. at a dilution of 1:100 in 5% BSA/PBS. After rinsing three times with PBS, the samples were incubated with Alexa Fluor 594 goat antimouse IgM secondary antibody (1:200; Thermo Fisher Scientific) for 30 min. in dark. Nuclei were counterstained with Hoechst dye at a dilution of 1:5000 for 15 min. at room temperature. Finally, coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA) mounting medium. Image acquisition was performed with a Zeiss Axiovert 35 (Carl Zeiss Microscopy GmbH, Jena, Germany) inverted fluorescent microscope with CellD (Olympus, Tokyo, Japan) software.