Ab290

Embryos were fixed for immunostaining for 20 minutes at room temperature in 4% PFA in phosphate buffer for 20 minutes, and all washes and incubations were performed in TBST + Ca²⁺ (50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl₂, 0.5% Triton X-100). Embryos were blocked in 10% donkey serum for 2 hours at room temperature; then, primary and secondary antibody incubations occurred in 10% donkey serum for 2 nights at 4 °C. Primary antibodies used include Developmental Studies Hybridoma Bank mouse IgG1 anti-Pax7 (1:10; PAX7) and mouse IgM anti-HNK-1 (1:5; 3H5); Novus Biologicals goat anti-FITC (1:500; NB600-493); Sigma rabbit anti-FLAG (1:100; F7425); Invitrogen Mouse IgG2a anti-V5 (1:500; R960-25); EMD Millipore rabbit anti-phosphohistone H3 (1:500; 06–570) and rabbit anti-Sox9 (1:1,000; AB5535); R&D Systems rabbit anti-cleaved caspase 3 (1:300; AF835) and goat anti-Sox10 (1:100; AF2864); Abcam rabbit anti-GFP (1:500; AB290); and Cell Signaling Technologies rabbit anti-pSmad1/5/8 (1:100; 13820) and rabbit anti-Snai2 (1:500; 9585). Primary antibodies were detected using Alexa Fluor 350/488/568/647-conjugated donkey secondary antibodies (1:500; Molecular Probes).

Following transcardial perfusion in 1× PBS, left brain hemispheres were fixed in 4% paraformaldehyde overnight (4°C) and processed using an Excelsior tissue processor (Thermo Fisher Scientific). Samples were then embedded in paraffin and sectioned at a thickness of 3 μm (Thermo Fisher Scientific, HM325). Immunostaining was performed as previously described (10 (link)). Briefly, following antigen retrieval, histological sections were blocked and permeabilized (5% donkey serum + 0.1% Triton X-100). Primary antibodies, anti-GFP (Abcam, ab290) and anti-HA (Cell Signaling Technology, clone C29F4: 3724S), to visualize AAV-transgene expression, were incubated on slides overnight (4°C). Alexa fluorophore-coupled secondary antibodies 488 and 568 (Molecular Probes) diluted in blocking buffer (5% donkey serum) were incubated on slides at room temperature for 1 hour before 4′,6-diamidino-2-phenylindole staining for 10 min. Slides were mounted for confocal imaging using an AxioScan microscope slide scanner (Zeiss).

Immunohistochemistry was generally performed as described previously [51 (link)] with an additional tissue section digestion by Proteinase K (50 µg/mL; Sigma-Aldrich P 6556) for 10 min. Then the sections were stained using an immunohistochemical staining kit (DakoCytomation Carpinteria, CA, USA, LSAB+ system-HRP, K0679) and either the anti-EGFP-antibody from Abcam (ab290) in a 1:800 dilution or from cell signaling (255S) in a 1:500 dilution.