Smi 32

Bielschowsky silver impregnation and Luxol fast blue periodic acid Schiff (LFB‐PAS) staining were performed according to standard procedures. Primary antibodies utilized for immunohistochemistry were against injured axons (APP, 1:2,000, clone 22C11, Chemicon) and nonphosphorylated neurofilaments (SMI32, 1:1,000, Covance, Princeton, NJ, USA), healthy phosphorylated neurofilaments/axons (SMI31, 1:10,000, Covance, Princeton, NJ, USA), myelin basic protein (MBP, 1:2,000, Dako), activated microglia (Mac3, also known as Lamp2, 1:200, clone M3/84, BD Pharmingen), glial fibrillary acidic protein (GFAP, 1:1,000, Dako), and foamy monocytes and macrophages (CD68, 1:5,000, clone KiM1P). Biotinylated secondary antibodies (GE Healthcare, Jackson ImmunoResearch and DCS Innovative diagnostic system), peroxidase conjugated avidin and DAB (Sigma‐Aldrich) were used for immunohistochemistry. Fluorescence labeled secondary antibodies (Cy3‐conjugated goat anti‐mouse IgG, 1:200, Jackson ImmunoResearch and Alexa488‐conjugated goat anti‐rabbit IgG, 1:200, Molecular Probes, Life technologies) were used for fluorescence immunohistochemistry.

Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with Triton 0.25%, and then blocked with 10% bovine serum albumin in 1× phosphate-buffered saline and 0.3% Triton X-100 for 1 h at room temperature. We incubated the cells with primary antibodies to NANOG (1:100, Abcam), SSEA-3 (1.100, Covance), ChAT (1:200, Chemicon & Millipore), SMI32 (Covance, 1:500), and Neurofilament-200 (1:1000 Sigma) overnight, and then with anti-rabbit or anti-mouse or -goat Alexa Fluor 488 or 594 (1:400; Life Technologies) secondary antibody for 1.5 h at room temperature.

To evaluate synaptic vesicles, retinal expression of synaptophysin was assessed. For fluorescence staining, samples were pre-embedded in 3% agar in deionized water. Vibratome sections (50 μm) were collected and washed several times in PBS. Sections were incubated in 10% normal donkey serum in PBS for 1 h at room temperature to block nonspecific binding activity, then with anti-rabbit synaptophysin (Cell signaling, Danvers, MA, USA) overnight at 4°C. After several washes with PBS, sections were incubated with goat anti-rabbit Alexa 546 (Molecular Probes). For double-labeling studies, sections were incubated with anti-mouse PKCα (Santa Cruz Biotechnology, Santa Cruz, CA), parvalbumin (Sigma, St. Louis, MO), calretinin (Millipore, Temecula, CA, USA), or SMI-32 (Covance, Emeryville, CA, USA) in 0.1 M PBS containing 0.5% Triton X-100 overnight at 4°C, rinsed for 30 min with 0.1 M PBS, and incubated with goat anti-mouse Alexa 488 (Molecular Probes) for 1 h 30 min at room temperature. After further washes in 0.1 M PB for 30 min, the sections were mounted using VectaShield Mounting Medium with DAPI (Vector Laboratories, H-1200). Slides were washed, covered with coverslips, and examined by confocal laser scanning microscopy (Zeiss LSM 510; Carl Zeiss Co. Ltd.).

The microfluidic devices were from Xona (SND 450). SMI-31, SMI-32 monoclonal antibodies were from Covance. EEA1, Rab5, Rab7 monoclonal antibodies were purchased from Cell signaling technology (CST), Lamp1 from DSCB, BIII-tubulin and MAP2 from Synaptic systems. Alexa Dye-conjugated goat secondary antibodies were from ThermoFisher.

Deeply anesthetized mice were perfused with 4 % PFA, and spinal cords with attached ventral roots dissected, post-fixed overnight, and cryoprotected in 30 % sucrose for 2 – 3 days at 4°C. After tissue embedding, 20 μm transverse sections of the spinal cord were cut on a cryostat and blocked with PBS containing 0.1 % Triton X-100, 5 % goat serum, and 0.5 % BSA, on glass slides. Sections were blocked with 5 % goat serum, incubated overnight with primary antibodies specific to PRG3 and nonphosphorylated neurofilament (SMI32, Covance) including 0.5 % BSA, washed, incubated with fluoro-chrome-conjugated secondary antibodies, and mounted in Fluoromount [52 (link)]. Images were acquired by confocal microscopy with an LSM510 microscope (Zeiss).