Ecl regent

Western blot analysis was carried out using an iBind Flex system (ThermoFisher Scientific) for primary and secondary antibody immunoblotting following a previously described method.^{29 (link)} For cell lysate collection SKOV3-ip1 and SKOV3-TRip2 cells were seeded in 6-well plates for 24 h at a density of 1.25 × 10⁵ mL⁻¹ and 1.75 × 10⁵ mL⁻¹, respectively, in 2 mL of media. Lysates were collected in RIPA buffer, protein quantified by Pierce BCA assay and 20–30 μg loaded on a 10% polyacrylamide gel which was run at 200 V. Rabbit monoclonal primary antibodies against anti-ALDH1A1 (Cell Signaling Technology) was used at a concentration of 1 : 1000 and anti-rabbit IgG secondary antibody (1 : 200 dilution; Cell Signaling Technology) for cell lysates analysis. Actin was used as a loading control for all experiments (1 : 1000 dilution; Cell Signaling Technology). After antibody incubations membranes were washed 5× with tris-buffered saline with tween (TBST) and visualised using ECL regent (GE Healthcare), with images taken using an iBright CCD camera (Invitrogen). Images were always acquired within the linear range of the camera to prevent the overexposure of any blots.