Mabn69

Mice were anaesthetized with Avertin and perfused intracardially with 4% paraformaldehyde in PBS. Dissected optic nerves and brain were post-fixed in 4% paraformaldehyde overnight at 4°C and 20μm frozen sections of optic nerve (longitudinal) and brain sections (coronal) collected. For immunohistochemistry, antigen retrieval was applied with reveal decloaker solution (Biocare Medical). Sections were incubated with primary antibodies overnight at 4°C. Primary antibodies include: MBP (SMI-99, 1:500, Covance); Olig2 (AB9610, Millipore, 1:250) and GFAP (MAB360, Millipore, 1:250); CC1 (OP80, Millipore, 1:250); Iba1 (019-19741, WAKO, 1:250); Caspr (MABN69, Millipore, 1:100); Na_v1.6 (ASC-009, Alomone Labs, 1:100) and K_v1.1 (APC-009, Alomone Labs, 1:100); Neurofilament 200 (N4142, Sigma, 1:250); Cre (#69050-3, Novagen, 1:250); NeuN (ab104224, Abcam, 1:250); Cdk5(sc-173, Santa Cruz, 1:200); NG2 (AB5320, Millipore, 1:250); Ki67 (#556003, BD pharmingen, 1:100). Primary antibodies were visualized with fluorescence-conjugated secondary goat anti-mouse IgG or goat anti-rabbit IgG FluoAlexa-594 or 488 antibodies (A-11032, A-11001, A-11008, A-11012, Invitrogen, 1:500). Nuclei were stained with DAPI (1:1000, sigma) and images collected using a Leica DFC500 fluorescence microscope.

Tissues (optic nerve, hippocampus, cortex and corpus callosum) were micro-dissected and homogenized with RIPA lysis buffer. Equal amounts of proteins were loaded and separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blocked in 0.1% Tween20 PBS buffer containing 5% BSA overnight at 4°C and incubated with primary antibodies followed by anti-mouse or anti-rabbit IgG conjugated to HRP. Enhanced chemiluminescence was performed with a West Pico Kit and detected by FluorChem E system (ProteinSimple, USA). The following primary antibodies were used: Cdk5 (sc-173, Santa Cruz, 1:1000); PLP (ab28486, Abcam, 1:1000); Caspr (MABN69, Millipore, 1:1000); MBP (SMI-99P, Covance, 1:1000); CREB (#9197, Cell signaling, 1:1000) and p-CREB (#9198, Cell signaling, 1:1000); Cofilin (#5175, Cell signaling, 1:1000) and p-Cofilin (#3311, Cell signaling, 1:1000); Fos (#4384, Cell signaling, 1:1000) and Arc (sc-17839, Santa Cruz, 1:1000) and β-actin (sc-47778, Santa Cruz, 1:1000). The density of immunoblots were quantified against normalized internal loading control using ImageJ software.