Rabbit anti gad67

Immunohistochemistry on 12 μm thick cryostat sections of lumbar level (L) 4 to 5 spinal cord was performed as previously described (Betley et al., 2009 (link)). Rabbit anti-βgal (gift from J. Sanes) (Gray and Sanes, 1991 (link)), rabbit anti-ChAT (generously provided by S.B.-M. and T.M.J., unpublished data), rabbit anti-GAD65 1:50,000 (Betley et al., 2009 (link)), mouse anti-GAD67 1:10,000 (Millipore), rabbit anti-GAD67 1:10,000 (Betley et al., 2009 (link)), chicken anti-GFP 1:1,000 (Millipore), sheep anti-GFP 1:1,000 (Molecular Probes), rat anti-NB2 (1A6) 1:4 (Shimoda et al., 2012 (link)), chicken anti-Pv 1:10,000 (generously provided by S.B.-M. and T.M.J., unpublished data), rabbit anti-RFP (Rockland), rabbit anti-Shank1a 1:64,000 (Betley et al., 2009 (link)), rabbit anti-Shank1a 1:1,000 (Millipore), mouse anti-Syt1 1:100 (ASV48, Developmental Studies Hybridoma Bank), and guinea pig anti-vGluT1 1:32,000 (Betley et al., 2009 (link)).

The 30 μm coronal cryosections were applied to glass slides and allowed to dry at room temperature overnight. Tissue sections were washed in phosphate-buffered saline (PBS) for 15 min and then blocked in 5% donkey serum, 1% BSA, and 0.2% Triton X-100 for 1 h at room temperature and incubated with primary antibody at 4 °C overnight. Mouse anti-pY99 (1:1000, Santa Cruz), rabbit anti-MAP2 (1:200, Protein Tech Co.), rabbit anti-pY416 (1:200, Cell Signaling), rabbit anti-pan Src antibody (1:200, Santa Cruz), rabbit anti-Dcx (1:200, [17 (link)], rabbit anti-Tbr1 (1:200, Abcam), rabbit anti-GAD67 (1:200, Millipore), and mouse anti-GM130 (1:200, BD) were used as primary antibodies. Donkey anti-species IgG conjugated with Alexa 488, Alexa 555 or Alexa 647, and Alexa 555-conjugated phalloidin were used as secondary antibodies. Hoechst 33342 (2 μg/ml, Molecular Probes) was used to visualize individual cell nuclei. Images were collected with a Zeiss LSM780 laser scanning confocal microscope (Advanced Fluorescence Imaging Core, SUNY Upstate Medical University). To compare the tyrosine phosphorylation response after different treatment, the average pY99 signal intensity was collected and normalized to the MAP2 immunosignal intensity and then compared among different treated group.