Pp n7927 00

Antibodies used are as follows: rabbit anti-GFP (1:500; Thermo Fisher Scientific, Invitrogen, A11122); rabbit anti-GFP polyclonal antibody, Alexa Fluor 488 (1:500; Thermo Fisher Scientific, no. A21311); rabbit anti-Cux1 (1:500; Santa Cruz Biotechnology, sc-13024 X); mouse anti-human Rorβ (1:300; Perseus Proteomics, PP-N7927-00); rabbit anti-Ctip2 (1:250; Abcam, ab240636); guinea-pig anti-Vglut1 (1:500; Merck Millipore, AB5905); mouse anti-NeuN (1:500; Chemicon, MAB377; clone A60); guinea pig anti-Vglut2 (1:1000; Sigma-Aldrich, Merck Millipore, AB2251-I); mouse anti-gephyrin (1:250; Synaptic Systems, no. 317005); mouse anti–synaptotagmin-2 (1:250; ZFIN, no. ZDB-ATB-081002-25); mouse monoclonal anti-PV (1:500; Sigma-Aldrich, Merck Millipore, no. p3088); goat anti-mouse Alexa Fluor 405 (1:200; Thermo Fisher Scientific, no. A-31553); goat anti-mouse Alexa Fluor 488 (1:500; Life Technologies, no. A-11029); goat anti-rabbit Alexa Fluor 488 (1:500; Life Technologies, no. A-11034); goat anti-rabbit Alexa Fluor 647 (1:500; Thermo Fisher Scientific, no. A-A21245); goat anti-mouse Alexa Fluor 647 (1:500; Thermo Fisher Scientific, no. A21236); and goat anti–guinea pig Alexa Fluor 647 (1:500; Thermo Fisher Scientific, no. A21450).

Organoids were fixed with 4% paraformaldehyde overnight at 4°C, cryoprotected in 30% sucrose solution, embedded in optimum cutting temperature (OCT) compound (Tissue Tek), and cryosectioned (16-μm thickness). After incubating with blocking buffer (PBS containing 5% goat or donkey serum and 0.3% Triton X-100) for 1 h at room temperature, sections were incubated overnight at 4°C with primary antibodies at the following dilutions: BRN3A (mouse, Chemicon, MAB1585, 1:500), CTIP2 (rat, Abcam, ab18465, 1:200), FOXG1 (rabbit, Takara, M227, 1:1000), LHX2 (goat, Santa Cruz Biotechnology, sc-19344, 1:1000), N-CADHERIN (mouse, Sigma, C3865, 1:1000), RAX (guinea pig, Takara, M229, 1:500), RORB (mouse, Perseus, PP-N7927-00, 1:200), SATB2 (rabbit, Abcam, ab34735, 1:200), SOX2 (goat, R&D, AF2018, 1:500), SOX10 (goat, R&D, AF2864, 1:200), TBR1 (rabbit, Abcam, ab31940, 1:200), TUBB3 (mouse, Sigma, T8660, 1:500). The samples were again washed three times with PBS and incubated with secondary antibodies conjugated with Alexa Fluor 488, Alexa Fluor 555, or Alexa Fluor 647 (Life Technologies) and Hoechst33342 (Dojindo Laboratories) for 1hat room temperature. After washing three times with PBS and once with distilled water, the preparation was mounted with ProLong Gold Antifade reagent (Thermo Fisher Scientific), and examined by using an LSM-710 confocal laser-scanning microscope (Carl Zeiss).