Ab5543

Polyethyleneimide 50%, borate buffer, N2 supplement, PBS, 4′,6′-diamidino-2-phenylindole dihydrochloride (DAPI), penicillin-streptomycin, paraformaldehyde (PFA), and laminin were purchased from Sigma-Aldrich. BrainPhys Neuronal Medium was purchased from STEMCELL Technologies. Rabbit anti-GFAP (1:2000; catalog #Z0334, Dako) with secondary antibody (1:200; catalog #AB6800, Abcam); mouse anti-B-III-tubulin (1:200; σT8660) with secondary antibody (1:200; catalog #21202, Thermo Fisher Scientific); rabbit anti-synaptophysin (1:100; catalog #ab32594, Abcam); and mouse anti-PSD96 (1:200; catalog #ab2723, Abcam) used the same secondary antibodies as above. Chicken anti-MAP2 (1:400; catalog #AB5543, Abcam) with secondary (1:200; catalog #A-16039, Thermo Fisher Scientific) and mouse anti-GAD65 (4 μg/ml; catalog #GAD-6-C, Developmental Studies Hybridoma Bank) with secondary (1:200; catalog #21202, Thermo Fisher Scientific) were used.

After CO generation, they were fixed, sectioned, and processed for immunofluorescence staining using previously reported protocols (Figure 2B,C) [29 (link),30 (link),31 (link),32 (link)]. Briefly, organoids were fixed in 4% paraformaldehyde, cyroprotected in 15% and 30% sucrose, embedded in OCT freezing medium, snap-frozen, and sectioned at 10–20 μm using a cryostat. The primary antibodies used for immunohistochemistry were PAX6 (rabbit, 1:250 dilution, Novus Biologicals Cat. #NB300-750, Littleton, CO, USA), MAP2 (chicken, 1:1000 dilution, MilliporeSigma Cat. #AB5543, Burlington, MA, USA), TBR1 (rabbit, 1:500 dilution, Abcam Cat. #AB31940, Cambridge, UK), and TBR2 (chicken, 1:200 dilution, MilliporeSigma Cat. #AB15894). Alexa Fluor secondary antibodies were used at a 1:500 dilution. Slides were mounted using Fluoromount G medium containing DAPI (4′,6-diamidino-2-phenylindole, SouthernBiotech, AL, USA). Images were obtained using a Zeiss 710 confocal microscope (Zeiss, Oberkochen, Germany). Fluorescent micrographs were processed using image processing software Fiji (NIH ImageJ, Version 1.53h, Bethesda, MD, USA).