Alexa fluor 488

Immunocytochemistry was performed on adherent EBs as previously described [16, 17] (link). Samples were xed with 4% paraformaldehyde, permeabilized with ice-cold methanol, and blocked using 3% bovine serum albumin (BSA) with 0.01% Triton X100 in phosphate-buffered saline (PBS). Samples were incubated overnight (at 4 o C in PBS with 1% BSA) with primary antibodies directed against α-actinin 2 (dilution 1:250; #701914, ThermoFisher Scienti c) or connexin 43 (dilution 1:200; #138300, ThermoFisher Scienti c). The samples were then incubated with a uorophore-conjugated secondary antibody (Alexa Fluor 488, dilution 1:5000; #715-546-150, Amersham or Cy3, dilution 1:1000; #711-166-152, Amersham) for 2 hours at room temperature. The samples were counterstained with Hoechst 33258 (0.5 µg/ml; Sigma, SA) and imaged on a Carl Zeiss LSM880 Airyscan™ confocal microscope (magni cation 40x) using ZEN software. Images were analysed using ImageJ (NIH, USA).

In the immunofluorescence analysis, cells were cultured on coverslips, fixed with 0.4% paraformaldehyde for 20 min and then permeabilized with 0.1% Triton X 100 for 10 min at room temperature. After being incubated at 37°C for 30 min with blocking solution, the cells were incubated overnight at 4 C with primary antibodies against GS or GPBB and then incubated for 1 h at room temperature with Alexa Fluor 488 (diluted 1:400; Amersham Biosciences, Amersham, UK).
Subsequently, the cells' nuclei were stained with 4 6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich, St. Louis, MO) for 10 min and then stored in the dark until they were evaluated. Finally, the stained cells were photographed with a confocal laser-scanning microscope (Leica TCS SP2 AOBS, Leica Microsystems, Tokyo, Japan). Primary antibodies against GS (diluted 1:100; Cell Signaling Technology, Danvers, MA, USA) and GPBB (diluted 1:100; Abcam, Cambridge, MA, USA) were used. All experiments were performed in triplicate.