Vectashield antifade mounting medium with dapi for nuclear staining

SCPCs were fixed in situ with 4% paraformaldehyde solution (ph7.4) for 15 min at room temperature, washed twice with (PBS) and then incubated with Alexa Fluor^® 488 Phalloidin (Life Technologies, USA) for filamentous actin (F-actin) according to manufacturer’s instructions. Chambers were dislocated from slides and coverslipped using Vectashield antifade mounting medium with DAPI for nuclear staining (VECTOR Laboratories Ltd. UK). Samples were visualised and imaged with a Zeiss confocal LSM700 or a Zeiss Axio Imager M2 upright microscope with fluorescence attachment.

To assess the purity of the cells, we performed immunofluorescence studies. All the steps, unless otherwise indicated, were performed at room temperature. Cultured cells were fixed in 4% buffered paraformaldehyde (PFA) for 10 minutes and then washed in PBS three times. After a common permeabilization step in 0.2% Triton X-100 in PBS for 10 minutes and an additional three washes in PBS, one set of samples was treated as follows: nonspecific binding was blocked by incubation in 10% normal goat serum for 1 hour and then incubated overnight at 4°C in rabbit anti-Islet-1 (Abcam, Cat. # ab20670, 2 μg/ml) or rabbit anti-GATA-4 (Abcam, Cat. # ab61170, dil. 1 : 500). After three additional washes in PBS, samples were incubated for 1 hour in FITC-conjugated goat anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA, Cat. # FI-1000, 10 μg/ml). Another set of samples was blocked in 10% normal rabbit serum for 1 hour and then incubated in goat anti-Nkx2.5 (Santa Cruz, Cat. # sc-8697, dil. 1 : 50) overnight at 4°C. Then, samples were incubated in AlexaFluor® 488-conjugated rabbit anti-goat antibody (Invitrogen, Cat # A-11078, dil. 1 : 200) for 1 hour. All the samples were then washed three times in PBS and mounted with VECTASHIELD® antifade mounting medium with DAPI for nuclear staining (Vector Laboratories, Cat. # H-1200).