A5228 clone1a1

Immunofluorescence staining was performed after fixation in 3% PFA 15 min at room temperature (RT). Permeabilization was obtained with Triton X-100 (Bio-Rad, Hercules, CA, USA) 1% for 10 min at RT and blocking solution composed of bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MI, USA) 2%, FBS 2%, Triton X-100 0.01% in PBS for 30 min at 37 °C was used for saturation of non-specific binding sites. Primary and secondary antibodies were incubated for 1 h at 37 °C while nuclei were counterstained with DAPI (Roche Diagnostic, Basel, Switzerland) at RT for 5 min before mounting the glass coverslips using DAKO mounting medium (Agilent, Santa Clara, CA, USA). The primary antibodies used here were LAP2 1:100 (611000, BD biosciences, Franklin Lakes, NJ, USA), ki67 1:200 (ab15580, Abcam, Cambridge, UK), α-SMA 1:200 (A5228 clone1A1, Sigma-Aldrich, St. Louis, MI, USA). The secondary antibodies used are: Alexa Fluor 488 anti-rabbit, 1:2000, and Alexa Fluor 568 anti-mouse, 1:1000 (Thermo Fisher Scientific, Waltham, MA, USA). Images were obtained with a confocal microscope (LSM900 Airyscan—Carl Zeiss, Oberkochen, Germany). Six (C) and ten (S) fields from two different strains of two different biological experiments were used for quantification.

rCEnC were plated on glass coverslip treated with FNC Coating Mix, in parallel to each cytofluorimetric analysis. After one rinse in DPBS, they were fixed in methanol for 10 min at − 20 °C, washed twice in PBS, and then preserved at 4 °C until staining. The non-specific binding sites were saturated with blocking solution composed of bovine serum albumin (BSA; Sigma-Aldrich, USA) 2%, FBS 2%, Triton X-100 0.01% in PBS for 30 min at 37 °C. Primary and secondary antibodies were diluted in blocking solution. Primary antibodies were incubated for 1 h at 37 °C, while incubation with secondary antibodies was done for 45 min at 37 °C. Nuclei were subsequently counterstained with DAPI (Roche, USA) 1:40,000 in PBS at RT for 5 min. Three rinses in PBS were performed between all steps except before incubation with the primary antibody. The glass coverslips were then dried and flattened on a glass slide using a DAKO mounting medium (Agilent, USA).
The primary antibodies used here were β-catenin 1:100 (ab32572, Abcam, UK), α-SMA 1:200 (A5228 clone1A1, Sigma-Aldrich, USA) and S100A4 1:100 (PA5-95736, Thermo Fisher Scientific, USA), while the secondary antibodies were Alexa Fluor 488 anti-rabbit, 1:2000, and Alexa Fluor 568 anti-mouse, 1:1,000 (Thermo Fisher Scientific, USA). Images were obtained with a fluorescent microscope (AXIO Imager.A1—Carl Zeiss).