Ab31940

Brain organoids were fixed in 4% paraformaldehyde for 45 min. After two washes in PBS, organoids were moved to 30% sucrose solution overnight at 4 °C. Organoids were then embedded in optimal cutting temperature (OCT) compound and flash frozen at –80 °C. Sections (20 µm) were cut with a cryostat (HM560 Microm Microtech). After permeabilization with 0.3% Triton and blocking with 2% goat serum (Gibco, 16210064) and 1% bovine serum albumin (BSA), sections were incubated for 1 hr with the following primary antibodies: SOX2 1:100 (Abcam, ab93689), PAX6 1:100 (Sigma-Aldrich, AMAb91372), βIII-tubulin 1:500 (Sigma-Aldrich, T8578), TBR1 1:100 (Abcam, ab31940), PSD95 1:500 (Sigma-Aldrich, MABN68), NeuN 1:100 (Sigma-Aldrich, MAB377), MAP2 1:100 (Sigma-Aldrich, ZRB2290), GFAP 1:200 (Sigma-Aldrich, G3893). After PBS washes, DAPI (100–1000 ng/ml) and secondary antibodies labeled with Alexa Fluor 488 or 594 1:500 (Invitrogen, #A-11037, #A-11029) were incubated for 1 hr. Slides were mounted using Fluoromount Aqueous Mounting Medium (Sigma Aldrich). Images were acquired using a microscope Axio Observer Z1 (Carl Zeiss), Objective 10 X, and analyzed using Fiji software.

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).

According to a previous article, briefly, cortical organoids were fixed in 4% paraformaldehyde overnight at 4 °C and then dehydrated by incubation with 30% sucrose overnight at 4 °C. The cortical organoids were then embedded in OCT compound (Sakura) and cryosectioned at 10 mm with a cryostat (Leica). For immunohistochemistry, frozen sections were washed with PBS before permeabilization with 0.2% Triton X-100 for 5 min at room temperature. The sections were blocked with 10% blocking serum (Solarbio, SL1) for 1 h at room temperature and then incubated with primary antibodies against the following in blocking solution at the corresponding dilutions: active caspase 3 (rabbit, 1:250, Abcam, ab32042), FOXG1 (rabbit, Abcam, ab196868, 1:500), NESTIN (mouse, Santa Cruz, sc-20978, 1:400), PAX6 (rabbit, BioLegend, PRB-278P, 1:300), SOX2 (rabbit, Cell Signaling, 3579, 1:400), TUJ1 (mouse, BioLegend, 801201, 1:500), PAX2 (mouse, Abnova, H00005076-M01, 1:400), CTIP2 (rat, Abcam ab18465, 1:500), TBR1 (rabbit, Abcam, ab31940, 1:200), vGLUT1 (mouse, Millipore, MAB5502, 1:400), GABA (rabbit, Sigma, A2052, 1:400), and ISL1 (mouse, Thermo, MA5-15515, 1:100). The secondary antibody used was Alexa Fluor 488- or 594-conjugated anti-donkey (1:500), and the sections were incubated with secondary antibody for 1 h at room temperature before being washed with PBS and subsequent microscopic inspection.