Apc 165

Dorsal and ventral spinal cord (Figure 1E) samples were microdissected from acute slices prepared in the same manner as done for electrophysiology (see below). Sample lysis was performed in RIPA buffer (Thermofisher) in the presence of protease and phosphatase inhibitors (Cell signaling). Samples concentration was determined with the Bradford method and protein migration and gel transfer was performed as described previously (Kenney and Rowitch, 2000 (link)). After blocking in Odyssey Blocking Buffer (PBS) (Li-Cor) for 1 hr, RT, primary antibodies were incubated O/N at 4°C onto the western blot membrane. The following antibodies were used: rabbit Kir4.1 (extracellular, APC-165, Alomone, 1:2000), rabbit Kir4.1 (APC035, Alomone, 1:2000), mouse GFAP (Sigma, 1:500), rabbit ALDH1L1 (ab87117,Abcam, 1:500), mouse β-actin (Sigma, A5441, 1:5000 or AC-74, 1:20000). IRDye Goat anti-mouse and anti-rabbit (680 and 800) fluorescent secondary antibodies (Li-Cor) were used for protein detection on the Odyssey Cxl imaging system.

The following antibodies were used for immunopanning, immunocytochemistry, immunohistochemistry and Western Blot experiments: goat anti-OLIG2 (AF2418, R and D Systems, 1:50), mouse anti-APC (clone CC1, OP80, 1:300, Millipore Sigma), mouse anti-NOGO-A (clone 11C7, gift from M.E. Schwab, 1:3,000), rat anti-MBP (ab7349, Abcam, 1:500), mouse anti-MOG (clone 8–18 C5, 1:1,000, Millipore Sigma), rat anti-GFAP (clone 2.2B10, 13–0300, Invitrogen, 1:1,000), rabbit anti-AQP4 (AB3594, 1:500, Millipore Sigma), rabbit anti-KIR4.1 (APC035, Alomone Labs, 1:3,000), rabbit anti-KIR4.1 (APC-165, Alomone Labs, 1:1,000), rabbit anti-KIR5.1 (APC123, Alomone Labs, 1:500), mouse anti-Neurofilament H (NF-H), nonphosphorylated (clone SMI32, 801701, Biolegend, 1:10,000), mouse anti-Neurofilament H (NF-H), phosphorylated (clone SMI312, 837904, Biolegend, 1:1,000), rabbit anti-IBA1 (019–19741, Wako, 1:500), goat anti-BRN3a (sc-31984, Santa Cruz, 1:200), rabbit anti-KCNQ2 (ab22897, Abcam, 1:200) rabbit anti-CASPR (ab34151, Abcam, 1:1,000), mouse anti-BRDU (347580, BD Biosciences, 1:200), rabbit anti-phospho-Histone H3 (pH3, 9701, Cell Signaling, 1:500), rat anti-CD140a (558774, BD Biosciences, 1:500), mouse anti-β-ACTIN (A5316, Sigma, 1:7,000).