Ab18465

Single and double immunohistochemical reactions were performed as described previously (Garcia-Moreno et al., 2012 (link)) using the following primary antibodies: Rabbit antibody to EGFP (Molecular Probes, A11122, 1:1000), mouse antibody to EGFP (Abcam, ab1218, 1:1000), chick antibody to EGFP (Aves GFP 1020, 1:10000), rabbit anti Nurr1 (Santa Cruz, sc-991, 1:400), rabbit antibody to Dbx1 (1:200; kind gift by Prof. Nakagawa, Univ. Minessota, United States), rabbit antibody to Tbr1 (Chemicon, AB9616, 1:1,000), rat antibody to Ctip2 (Abcam, ab18465, 1:500), rabbit antibody to dsRed2 (Takara, 632475, 1:1000), and rabbit antibody to Pax6 (Covance, PRB-278P, 1:200). Dbx1 immunostaining required antigen retrieval with citrate acid.
For secondary antibodies (all 1:1000), we used Alexa 568 goat antibody to rabbit IgG (Molecular Probes, A11011), Alexa 647 goat antibody to rabbit IgG (Molecular Probes, A21245), Alexa 488 goat antibody to rabbit IgG (Molecular Probes, A11034), Alexa 488 goat antibody to mouse IgG (Molecular Probes, A11001), Alexa 568 antibody to mouse IgG (Molecular Probes, A11004), Alexa 568 goat antibody to rat IgG (Molecular Probes, A11077), and Alexa 488 goat antibody to chicken (Invitrogen, A11039).

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).