Zeissimager z1axiocammrm

The E, M and RE cells were incubated with 1 μL of BrdU solution (ThermoFisher, Waltham, MA, USA) for 90 min, washed with a phosphate-buffered saline solution (PBS) and fixed using 4% formaldehyde (Sigma-Aldrich, Burlington, VT, USA). Next, the cells were treated with 2M-HCl (Merck, Burlington, VT, USA), washed with PBS-0, 5% Tween 20–0 and 5% BSA (Sigma-Aldrich, Burlington, VT, USA) and incubated with anti-BrdU antibody (1:10, ThermoFisher, Waltham, MA, USA) and anti-mouse Ig-FITC (1:100, ThermoFisher, Waltham, MA, USA). The coverslips were mounted using Vectashield-DAPI mounting medium (Vector Laboratories, Burlingam, CA, USA). Images were taken with ZeissImager.Z1AxioCamMRm (Zeiss, Oberkochen, Germany), and the stained nuclei were counted. Statistical analysis was performed using the Mann–Whitney test [28 (link)].

The E, M and RE cells were fixed with methanol (Merck, Burlington, VT, USA), blocked using 3%BSA-PBS-0.5%Tween20 (Sigma-Aldrich, Burlington, VT, USA) and incubated with anti-Snail (L70G2, 1:50, Cell Signaling, Danvers, TX, USA), anti-MMP2 (42-5D11, 1:50, Calbiochem, San Diego, CA, USA) and anti-mouse Alexa 488 (A-11029, 1:500, ThermoFisher, Waltham, MA, USA). For E-cadherin and Fibronectin co-immunocytochemistry, the cells were fixed and blocked as previously described and co-incubated with anti-E-cadherin (1:50, Cell Signaling, Danvers, TX, USA), anti-Fibronectin (1:50, Santa Cruz, Dallas, TX, USA) overnight at 4 °C and anti-rabbit or anti-mouse Alexa 488 or 594, respectively, at room temperature for 1 h (1:500, ThermoFisher, Waltham, MA, USA). Coverslips were mounted using Vectashield-DAPI mounting medium (Vector Laboratories, Burlingam, CA, USA). Images were taken with ZeissImager.Z1AxioCamMRm (Zeiss, Oberkochen, Germany). These experiments were repeated at least three times, where several fields were photographed to represent the maximum intensity projection.