Goat anti mouse alexa fluor 555 antibody

For the analysis of formation and repair of DNA double-strand breaks during and after irradiation, 50,000 MMTV-PyMT WT or K_Ca3.1 KO cells were seeded for 72 h in twelve-well chambers (ibidi, Planegg, Germany) in triplicate. TRAM-34 or its solvent was applied 1 h prior to irradiation with a single fraction of 0, 2, 4, or 6 Gy. After further 30 min or 24 h of culture, cells were fixed with 70% ethanol at −20°C for 10 min. Then, the cells were washed twice with 250 µL PBS and unspecific epitopes were blocked with 10% normal goat serum (Linaris, Dossenheim, Germany) in PBS for 1 h. Blocking solution was removed and the cells were incubated for 2 h in a primary mouse anti-γH2AX antibody (613402, BioLegend, London, UK) solution (1:500 dilution in 1.5% normal goat serum/PBS). Next, cells were washed twice with PBS and incubated with a secondary Alexa Fluor^® 555 goat anti-mouse antibody (A21127, Thermo Fisher Scientific) diluted 1:800 in 1.5% normal goat serum in PBS for 1 h. After two final PBS washing steps, the silicone chamber walls were removed, and the slide was cover-slipped with DAPI-containing vectashield solution. Four micrographs of different areas were taken using the F-view II camera (SIS, Munster, Germany) attached to fluorescence microscope (Olympus, Hamburg, Germany) and cell nuclei as well as the number of γH2AX foci per cell were counted.

After stimulation, cells were fixed with 4% paraformaldehyde for 5 min at room temperature, permeabilized with 0.01% Triton X-100 for 5 min and blocked with 5% BSA in PBS for 20 min. Specimens were incubated with primary mouse anti-β-catenin (1:500, clone CAT-5H10, Thermo Fisher Scientific, Waltham, MA, USA) and an anti-αSMA antibody (1:200, clone 1A4, Biolegend, San Diego, CA, USA) at room temperature, followed by the secondary AlexaFluor555 goat anti-mouse antibody (1:8000) and phalloidin (1:1000, all Thermo Fisher Scientific) at room temperature for 1 h. Hoechst (1:1000, Thermo Fisher Scientific) was used to label the nuclei. Immunofluorescence was analyzed using Leica DM5500 B fluorescence microscope with DFC365 FX camera (Leica Microsystems, Wetzlar, Germany).