P75ntr

As previously described, every fifth sagittal section was immunochemically stained for subsequent fluorescent microscopic analysis using a double labeling procedure involving monoclonal and polyclonal antibodies [15 (link)]. These antibodies were raised against the low-affinity neurotrophin receptor (p75^NTR; 1 : 5,000; Abcam, Cambridge, MA), serotonin (5HT; 1 : 5,000; Immunostar, Hudson, WI), or calcitonin gene related peptide (CGRP; 1 : 1,000; Peninsula Laboratories, San Carlos, CA) for identifying the implanted SCs or delineating the growth of specific descending or ascending axonal populations, respectively, within the p75⁺ SC implants. Sections were then washed and incubated with corresponding fluorescent secondary antibodies (Alexa 488- or Alexa 594-conjugated goat anti-rabbit or anti-mouse antibodies, 1 : 200; Molecular Probes, Eugene, OR). Sections were mounted onto Snowcoat X-tra slides (Surgipath, Richmond, IL) and cover-slipped with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) containing the nuclear dye Hoechst for storage at 4°C.

Schwann cells were harvested and purified according to O’Neill et al.^{13 (link)}, modified from Raff et al.^{64 (link)}. Briefly Schwann cells were purified from P7 sciatic nerves of F344‐Tg(UBC‐EGFP)F455Rrrc (RRRC, Rat Resource and Research Centre) and cultured in DMEM with 5% FBS and supplemented with 0.5 μM forskolin (Sigma-Aldrich) and 50 nl/ml heregulin β1 (R&D Systems). Schwann cells were purified using an immunomagnetic positive selection kit (EasySep, Stem Cell Technologies), with the antibody p75^NTR (Abcam).

For immunocytochemistry, OECs and microglia were washed with PBS, and fixed in 4% PFA for 20 min at room temperature. After blocked with 3% BSA for 15 min, cells were stained with primary antibodies: P75NTR (ab52987, 1:500; Abcam), iNOS (ab49999, 1:200; Abcam), F4/80 (SAB5500103, 1:100; Sigma), p‐c‐Jun (3270s, 1:5000; Cell Signaling Technology), and p‐NF‐κB (3033t, 1:2000; Cell Signaling Technology) at 4°C overnight. The cells were then stained with corresponding secondary antibodies. A confocal microscope (LSM 800; Zeiss) was utilized for examination and photography.
For immunohistochemistry, slides of injured spinal segments were blocked in 0.01 M PBS containing 3% BSA and 0.1% Triton X‐100 for 20 min. Subsequently, slides were incubated with primary antibodies: Iba1 (019‐19741, 1:1000; Wako), iNOS (ab49999, 1:200; Abcam), Arginase1 (sc‐166920, 1:200; Santa Cruz Biotechnology), GFAP (1:2000; Sigma), NeuN (#24307, 1:200; Cell Signaling Technology) at RT overnight. The slides were then stained with corresponding secondary antibodies, and counterstained with Hoechst 33342 to display nuclei. Slides were photographed by a confocal microscope (LSM 800; Zeiss).

Cells were fixed in 4% PFA for 15 minutes followed by two 1xPBS washes. Cells were blocked by 10% bovine serum in 1xPBS at room temperature for 1 hour. Primary antibodies were applied for overnight at 4°C in 1xPBS containing 1% BSA and 0.25% Triton-X-100. Following three washes, alexa fluor conjugated secondary antibodies (1:1000, invitrogen) together with DAPI (1:2000) were added for 1 hour. After three more washes, coverslips were mounted with Prolong Gold antifade reagent (Invitrogen) and imaged. Primaries antibodies used were: Oct4 (1:1000, Santa Cruz), Sox2 (1:1000, Millipore), Ssea4 (1:500, Chemicon), Tra1-60 (1:500, Chemicon), Collagen type IV (1:500, Abcam), Gata4 (1:1000, Santa Cruz), Map2 (1:1000, Abcam), Gli1 (1:500, Santa Cruz), Forse1 (1:300, DSHB), Nkx2.1 (1:500, Abcam), Pax6 (1:1000, DHSB), Mash1 (1:1000, BD Pharmingen), p75-NTR (1:1000, Abcam), VAChT (1:1000, SYSY), HB9 (1:1000, Abcam) and ChAT (1:600, Aves Labs).