Research inverted system microscope

After culture for 7 days, the prevascularized cell sheets (iEC/UM) were washed with phosphate-buffered saline (PBS), fixed in 4% paraformaldehyde for 15 min, and then blocked in a 5% goat serum-PBS buffer solution for 1 h at room temperature. A primary antibody rabbit anti-mouse CD31 (ab124432, Abcam, dilution 1:500) in 1% bovine serum albumin (BSA)-PBS was added to the samples and incubated overnight at 4°C. After washing with PBS, a secondary antibody goat anti-rabbit (Alexa Fluor 594, 2 μg/mL, Invitrogen) in 1% BSA-PBS buffer was added and incubated in the dark for 1 h at room temperature. Finally, the fluorescent staining images were captured by confocal microscopy (Research inverted system microscope, IX71, Olympus).

Immunohistochemistry and immunofluorescence were performed as reported (Sundaram et al., 2012 (link); Chen et al., 2017 (link)). In brief, mice were deeply anesthetized by 2% pentobarbital sodium and transcardially perfused with 4°C PBS and 4% paraformaldehyde. Brains were extracted and fixed in 4% paraformaldehyde for 24 h; 4 μm sections of the midbrain and the striatum were sliced by Leica RM vibratome (Leica Microsystems, Heidelberg, Germany). Slices for immunohistochemistry were incubated with anti-TH antibody (Millipore, Billerica, MA, United States). Goat against rabbit secondary antibody was from ZSGB-BIO (Beijing, China). Slices for immunofluorescence were incubated with anti-TH (Millipore, Billerica, MA, United States) or anti-GFP antibody (Aves Labs, Washington, DC, United States). Secondary antibodies were goat against rabbit antibody (Alexa Fluor^® 594) and goat against chicken antibody (Alexa Fluor^® 488). The nucleus was stained by DAPI (Boster, China). Images were obtained by research inverted system microscope (Olympus, Tokyo, Japan) or laser scanning confocal microscope (Olympus, Tokyo, Japan). The gray level was analyzed by ImageJ (National Institutes of Health, Bethesda, MD, United States) and the number of TH positive neurons was counted blindly by independent investigators.