Quantitative Immunofluorescence Analysis of EMT and PARP-1

To analyze the protein expression of EMT markers and PARP-1 was performed the immunofluorescence assay according to Gelaleti et al. (2017) [17 (link)]. Briefly, 6 × 10⁴ cells of CF41 and MDA-MB-468 cell lines were transferred to a silicone separator attached to a slide and kept in the incubator for 24 h at 37 °C with 5% CO₂. Then, the cells were treated with carboplatin, losartan, or both in combination. For the immunofluorescence technique were used the specifics primary antibodies E-cadherin (1:200) (catalogue number 3195S, Cell Signaling Technology, Danvers, MA, USA), N-cadherin (1:100) (catalogue 7939, Santa Cruz Biotechnology, Dallas, TX, USA) and PARP-1 (1:50) (catalogue 436400, Thermo Fisher Scientific, Waltham, MA, USA) and the secondary antibody used was Alexa Fluor 648 anti-mouse IgG (1:100) (Sigma-Aldrich). To analyze the protein expression, the cells were captured using a microscope containing a specific software (OLYMPUS, model BX53, software Image-Pro Plus version 7.0, Rockville, MD, USA). All experiments were carried out in triplicate and protein expression was quantified according to Jardim-Perassi et al. (2014) [18 (link)] as cited by Gelaleti et al. (2017) [17 (link)]. The analyses were performed using ImageJ Software (NIH, Bethesda, MD, USA) and all values were obtained in arbitrary units (a.u.) and represented as the mean optical density (M.O.D.).