Smi 312r

For immunofluorescence, we used rabbit anti-C3G (Santa Cruz Biotechnology, H-300/sc-15359, 1:150), mouse anti-N-Cadherin (Abcam # ab98952, 1:200), mouse anti-Nestin (BD Biosciences #611658, 1:200), rabbit anti-NF medium chain (Abcam #ab64300, 1:200) or mouse anti-NF medium chain (2H3, DSHB, 1:4), rabbit anti-NF light chain (Cell Signaling #2837, 1:200), mouse anti-SMI 312 (Covance SMI-312R, 1:200), rabbit anti-Cux1 (Santa Cruz Biotechnology #sc13024, 1:150), rabbit anti-Tbr1 (Abcam #31940, 1:400), rabbit anti-Tbr2 (Abcam #23345, 1:400), rat anti-β1 integrin [very late antigen (VLA), Chemicon, #MAB1997, 1:200], rabbit anti-Calretinin (Millipore #AB5054, 1:1000), mouse anti-chondroitin sulfate (clone CS-56, Sigma #C8035, 1:100), rabbit anti-Tbr1 (Abcam #ab31940, 1:500), mouse Tau-1 (Chemicon #MAB3420; 1:500), mouse anti-MAP2 (Chemicon #AB5622; 1:1000), Hoechst 33342 (Molecular probes, 1:6000) and goat secondary antibodies labeled with Alexa 488 or 594 (Molecular Probes, 1:800). The rat 9EG7 (supernatant) antibody was provided by D. Vestweber (Max Planck Institute for Molecular Biomedicine, Münster, Germany). Images were taken on a Zeiss LSM 700 confocal microscope using the Zeiss ZEN software.

Cells were fixed in 4% PFA in Pagano solution (250 mm sucrose, 25 mm MgCl₂, 2.5 mm KCl, 25 mm HEPES, pH 7.4; + phosphatase inhibitors for phospho immunostaining) for 15 min at room temperature, rinsed 3× for 5 min in Pagano, and incubated with blocking solution (Pagano + 0.25% Saponin + 2% BSA) for 1 h. Cells were incubated in primary antibodies diluted in blocking solution for 1 h, rinsed 3× for 5 min with Pagano solution, incubated with appropriate secondary for 1 h, then rinsed 3× for 5 min with Pagano solution. The primary antibodies used were anti-MAP2 (1:250, Chemicon, ab5756), anti-SMI-312 (1:1000, Covance, SMI312R), anti-p-Akt (1:100, Santa Cruz, sc-514032), Cat-315 (1:20, from R. Matthews; Dino et al., 2006 (link)), CS56 (1:200, Abcam, ab11570), and anti-DCX (1:500, gift from C. Walsh; Gleeson et al., 1999 (link)) polyclonal sera. Appropriate Alexa Fluor 488-conjugated, Alexa Fluor 555-conjugated, and Alexa Fluor 647-conjugated secondary antibodies were used. Hoechst 33342 (2 μg/ml; Invitrogen) was used to counterstain nuclei. Immunohistochemistry for explants was performed as previously described (Nichols et al., 2013 (link)). Images were collected with a Zeiss LSM780 laser scanning confocal microscope (SUNY Upstate Advanced Fluorescence Imaging Core).