Alexa fluor 555 goat anti mouse secondary antibody

Differentiated C2C12 cells were grown on cover slips and fixed with ice cold acetone for 10 min at -20 °C. After fixation, cells were washed in PBS with 5min per time for three times and then blocked with 10% normal goat serum for 2 hours. Primary antibody against myosin heavy chain (#MF20, DSHB, Iowa City, IA, USA) was diluted in 10% normal goat serum (1:200) and cells were incubated with primary antibody for overnight at 4 °C. After incubation, cells were washed three times in PBS for 5 min at a time and incubated with Alexa Fluor 555 goat anti mouse secondary antibody (#A32727, ThermoFisher Scientific, Waltham, MA, USA) for 2 hours at room temperature. Cells were washed three times and counterstained with DAPI. After washing, cover slips were mounted with mounting media. Images were obtained with Keyence fluorescent microscopy (Osaka, Osaka, Japan).

The expression of cytokeratin-18 (CK-18) was detected by immunofluorescence to confirm the epithelial GEC phenotype. 4 × 10⁴ cells/wells were cultured on pre-coated collagen type I on sterile glass microscopic slides and incubated at 37 °C, 5% CO₂ for 24 h. Then, cells were fixed with 4% paraformaldehyde in PBS for 15 min at RT, washed three times in PBS 1X and permeabilized in 0.02% Triton X-100 for 20 min. GECs were blocked with 5% BSA and 2% goat serum in PBS for 1 h at RT. Thereafter, GECs were incubated at RT for 1 h using the CK-18 primary antibody (sc- 32329-Santa Cruz Biotechnology Dallas, TX, USA), diluted 1:100 in PBS with 5% BSA, washed three times and then incubated at RT for another hour with the Alexa Fluor 555 goat anti-mouse secondary antibody (A-21422, Thermo Fisher Scientific, USA), diluted 1:500 in PBS with 5% BSA. After washing the slides three times in PBS 1X, the samples were treated using the UltraCruz Aqueous Mounting medium with DAPI (sc-24941, Santa Cruz Biotechnology Dallas, TX, USA). Human epithelial cell lines, known as HeLa cells (ATCC® CCL-2), and human embryonic kidney 293 T cell lines (ATCC® CRL-3216) were used as positive and negative controls, respectively. The slides were analyzed using the EVOS FL Cell Imaging Station (Thermo Fisher Scientific, USA).

RPMI 1640 medium and DMEM (Dulbecco’s Modified Eagle Medium) were acquired from Capricorn Scientific GmbH (Ebsdorfergrund, Germany). L-glutamine, trypsin, and penicillin–streptomycin solution were from Biowest (Nuaillé, France). Thiazolyl blue tetrazolium bromide (MTT), fetal bovine serum (FBS), DMSO, Hoechst 33342, and carboxyfluorescein succinimidyl ester (CFSE) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). An Apoptosis Detection Kit based on Annexin-V-FITC (AV) and propidium iodide (PI) staining was acquired from Abcam (Cambridge, UK). Bovine serum albumin (BSA) was obtained from Serva (Heidelberg, Germany). Rhodamine 123 (Rho123) and tetramethylrhodamine ethyl ester (TMRE) were purchased from Sigma (St Louis, MO, USA). Carbonyl cyanide m-chlorophenyl hydrazine (CCCP, C2759) was purchased from Sigma-Aldrich (Darmstadt, Germany). FITC-conjugated anti-P-gp antibody was obtained from BD Biosciences (Winnersh, Berkshire, UK). Anti-P-gp mouse monoclonal antibody (Abcam, Cambridge, UK; ab10333) and secondary antibodies Alexa Fluor 555 goat anti-mouse secondary antibody (#A-21422, Thermo Fisher Scientific, Waltham, MA, USA) were obtained.