Revolution xd confocal laser scanning microscope

At room temperature, cardiac tissues were fixed inside the chips with 4% paraformaldehyde for 1 ​h. For immunostaining, tissues were embedded at optimal cutting temperature and cryo-sectioned to 20 ​μm thickness. After permeabilization (0.2% Triton X-100), 3% bovine serum albumin (BSA) was used to block the samples. The following primary antibodies were used for immunostaining: mouse anti-α-actinin (Sarcomeric) (Sigma-Aldrich SAB4200813; 1:200); mouse anti-cTnI (Sigma-Aldrich MFCD01864286; 1:200); rabbit anti-connexin-43 (Cell Signaling 3512 ​S; 1:75); and the proper secondary antibodies: Alexa Fluor 594 goat anti-mouse IgG (Invitrogen A-21044, 1:200); Alexa Fluor 488 goat anti-mouse IgG (Invitrogen A-10680, 1:200); Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen A-11037, 1:200). The nucleus was stained with DAPI (Beyotime, 1:200). A Revolution XD confocal laser scanning microscope (Andor) was used for imaging.

After induction of RA and/or miR-218 for a period of time, ASCs were subjected to immunofluorescent staining by rabbit anti-rat βIII-Tubulin primary antibody to detect differentiated neural cells. After washing with PBS, the samples were incubated with the appropriate secondary antibody, mouse anti-rabbit Alexa-Fluor 647 (1:200 in 1% BSA; Cell Signaling Technology) in the dark. Then samples were washed twice with PBS, the nuclei stained with 10 μg/mL Hoechst 33342 (HOE, Sigma Aldrich,) for 0.5 h, and images were obtained using a Revolution XD confocal laser scanning microscope (Andor, Belfast, Northern Ireland).