Antifate mounting medium containing dapi

Cells were seeded on Gelatin-coated coverslips and treated with doxorubicin for 20 hours. After treatment, cells were washed with 3 X PBS, fixed with 4% parafomaldehyde (15 mins), washed permabilized with 0.2% Triton-X (15 mins), washed and blocked for 3 hours in 3% goat serum and incubated with γ-Η2Α.X (Abcam) overnight. Cells were washed with 3 X PBS and incubated for one hour in secondary antibody Alexa Fluor-488 (Abcam) and were then washed and mounted on a slide using Antifate mounting medium containing DAPI (VECTASHIELD, VECTOR laboratories). Coverslips were viewed on Zeiss LSM880 confocal microscope. A minimum of three images were captured from each sample and were quantified using ImageJ based on fluorescence intensity of γ-Η2Α.X staining per nucleus. Graphs show intensity relative to vehicle-only treated cells with a minimum of three independent biological replicates.

Cells were seeded on Matrigel-coated coverslips and were left to attach for 2 days. Cells were washed with 3 X PBS, fixed with 4% parafomaldehyde (15 mins), washed and permabilized with 0.2% Triton-X (15 mins), washed and blocked for 3 hours in 3% goat serum and incubated with cTnT (Abcam) and sarcomeric α-actinin (Abcam) overnight. Cells were washed with 3 X PBS and incubated for one hour with secondary antibody Alexa Fluor-563 (Abcam) and Alexa Fluor-488 (Abcam) and were then washed and mounted on a slide using Antifate mounting medium containing DAPI (VECTASHIELD, VECTOR laboratories). Slides were viewed on Zeiss LSM880 confocal microscope.