Rabbit anti pax6

Brains were fixed overnight in 4% paraformaldehyde (PFA) at 4°C, followed by submersion in 30% sucrose until sinking, as previously described (Silver et al. 2010 (link)). Brain cryostat sections (20 µm) were prepared and stored at −80°C until use. Sections were permeabilized with 0.25% Triton X-100 for 10 min and blocked with MOM block reagent (Vector laboratories) for 1 h at room temperature (RT). Sections were incubated with primary antibodies for 2 h at RT or overnight at 4°C. Sections were then incubated in species appropriate secondary antibodies and Hoechst for 15 min at room temperature. The following primary antibodies were used: rabbit anti-CC3 (diluted 1:200; Cell Signaling); rabbit anti-TBR2 (1:1,000; Abcam); rabbit anti-PAX6 (1:1,000; Millipore); mouse anti-TUJ1 (1:400; Covance). The following secondary antibodies were used: Alex Fluor 488 and Alex Fluor 594 (1:400; Invitrogen). Hoechst (Thermo Fisher Scientific) was used for nucleus counterstain. High-magnification images were captured using a Zeiss Axio Observer Z.1 microscope coupled with an apotome. Cortical thickness was measured with Zen software. Cell quantification was performed with ImageJ/FIJI. Three sections from anatomically comparable regions per embryo and three biological replicates from control (wild-type) and mutant alleles (Casc3^RRU345/+, Casc3^{RRU345/RRU345}, and Casc3^RRU345/Null) were quantified.

Fluorescence immunohistochemistry utilized a citrate-based antigen retrieval step involving pH 6.0 sodium citrate in water heated to 95 °C for 7 min, followed by cooling to room temperature and immunostained as described previously (38 (link)). Antibodies and dilutions are as follows: rabbit anti-Pax6 (Millipore; 1:750), rabbit Hopx (Sigma; 1:500); mouse anti-Ki67 (Vector; 1:300), goat anti-EGFR (R&D Systems; 10 mg/mL); rabbit anti-Olig2 (Abcam; 1:2,000), rabbit anti-Tbr1 (Abcam; 1:1,000), rat anti-GFAP (1:100), mouse anti-βIII-tubulin (R&D Systems; 1:1,000), chicken anti-Tbr2 (Millipore; 1:200), rat anti-BrdU (Accurate Chemical; 1:400); secondary antibodies and dilutions were donkey anti-goat/chicken/rat, 1:1,000, DyLight 488/543/647 (Jackson Laboratories). Blocking utilized reconstituted Donkey Serum. Sections were counterstained with DAPI mounting medium (Vector) to label cellular nuclei. A single injection of BrdU at 50 mg/kg (Sigma; in 0.9% NaCl with 0.007 N NaOH) was delivered intravenously at E97, with sacrifice 1 h later.

The following primary antibodies and dilutions were used for immunohistochemistry (IHC) staining and western blotting: rabbit polyclonal anti-UTX (1:1,000; Millipore, #ABE409); mouse monoclonal anti-SOX2 (1:500; R & D, #MAB2018); rabbit anti-PAX6 (1:1,000; Millipore, #AB2237); rabbit anti-TBR2 (1:1,000; Abcam, #AB23345); mouse monoclonal anti-β-ACTIN (1:20,000; Proteintech, #60008-1-Ig); rat monoclonal anti-BrdU (1:1,000; Abcam, #AB6362); rabbit monoclonal anti-Ki67 (1:1,000; Abcam, #AB15580); rabbit anti-PCNA (1:500; Santa Cruz Biotechnology, #SC7907), rabbit anti-TUJ1 (1:1,000; Sigma, #T2200); rabbit monoclonal anti-PTEN (1:1,000; Cell Signaling Technology, #9188); rabbit monoclonal anti-AKT (1:1,000; Cell Signaling, #4685); rabbit monoclonal anti-phospho-AKT (1:1,000; Cell Signaling, #3787); rabbit monoclonal anti-mTOR (1:1,000; Cell Signaling, #2983); rabbit monoclonal anti-phospho-mTOR (1:1,000; Cell Signaling, #5536); rabbit monoclonal anti-H3 (1:4,000; Cell Signaling, #4499S); rabbit polyclonal anti-trimethyl-histone H3 (Lys27) (1:2,000; Cell Signaling, #3377S); and rabbit anti-FLAG (1:1,000; Sigma, #7425).