Neurofilament

Dorsal root ganglion cell culture after electrical stimulation and serial 8-mm-thick section of nerve, muscle, dorsal root ganglion cells, and the brain were cut using a cryostat, and mounted on superfrost/plus slides (Menzel-Glaser, Braunschweig, Germany) were subjected to immunohistochemistry using antibodies against NGF-R (1:1000, Abbiotec), S-100(1:200, Serotec), neurofilament(1:200, Millipore), anti-synaptophysin (Abcam, 1:200 dilution), and anti-TNF-α (Abcam, 1:300 dilution) to detect the inflammatory response associated with nerve regeneration in sciatic nerve, dorsal root ganglion cells, and the brain. The immunoreactive signals were observed using AF 488 donkey anti–mouse IgG and AF594 donkey anti-rabbit (Invitrogen; 1:200 dilutions) and were then viewed using an Olympus BX40 Research Microscope.

The antibodies used were as follows: vGluT2 (1:1,000, Cat. AB2251, Millipore, Billerica, MA, USA), neurofilament (1:5,000, Cat. NFH, Aves Labs, Tigard, Oregon), MBP (1:1,000, Cat. SMI-99, Sternberger Monoclonals), synaptophysin 1 (1:1,000, Cat. 101 011, Synaptic Systems, Gottingen, Germany), Olig2 (1:250, Cat. 18953, IBL Co., LTD, Japan) and NG2 chondroitin sulfate proteoglycan (1:500, Cat. MAB5384, Chemicon International, Billerica, MA, USA).

Five micrometer-thick paraffin sections were immunostained at room temperature for 1 h with antibodies against rat AQP4 (1:200, Santa Cruz Biotechnology), GFAP (1:100, Millipore), myelin basic protein (MBP) (1:200, Santa Cruz Biotechnology), ionized calcium-binding adaptor molecule-1 (Iba1; 1:1,000; Wako), albumin (1:200, Santa Cruz Biotechnology), Ly-6G (1:100, Santa Cruz Biotechnology), C5b-9 (1:50, Hycult Biotech), CD45 (1:10, BD Biosciences), CD163 (1:50, Bio-Rad Laboratories), neurofilament (1:200, Millipore), iNOS (1:100, BD Biosciences) or arginase-1 (1:50, Santa Cruz Biotechnology), followed by appropriate fluorescent secondary antibody (1:200, Invitrogen) or biotinylated secondary antibody (1:500, Vector Laboratories). Tissue sections were examined with a Leica (Wetzlar, Germany) DM 4000 B microscope. AQP4, GFAP and MBP immunonegative areas were defined by hand and quantified using ImageJ. Data are presented as area (mm²) of immunonegative area.

After organotypic culture, SGN explants were incubated in ice-cold PBS at 4°C for 30 min and washed twice with PBS. In order to keep the architecture structure of SGNs, the coated matrigel was carefully removed except that the surrounded cultured tissue, followed by the fixation with 4% paraformaldehyde and permeabilization with 1% TritonX-100 in PBS (Sigma), samples were immersed in blocking solution (0.1% TritonX-100, 8% donkey serum, 1% bovine serum albumin, and 0.02% sodium azide in PBS) at room temperature for 1 h. Then, samples were incubated with different primary antibodies: β-tubulin (1 : 1000, Neuromics, USA), neurofilament (1 : 1000, Millipore, USA), and synaptophysin (1 : 1000, Millipore), diluted in blocking solution, respectively, at 4°C overnight. The next day, tissues were incubated with FITC-conjugated or TRITC-conjugated (1 : 1000, Invitrogen) secondary antibody along with DAPI (1 : 800, Sigma-Aldrich) or phalloidin (1 : 1000, Sigma-Aldrich) in 0.1% TritonX-100 and 1% BSA in PBS at room temperature for 1 h. Then, the coverslips were mounted and observed under a laser scanning confocal microscope (Leica, Germany).

ICC analyses were performed to evaluate the expression of genes related to pluripotency and differentiation. Before staining, all cell samples were preincubated for 10 min at 4°C and fixed with 4% paraformaldehyde for 30 min. After washing twice with Dulbecco’s phosphate-buffered saline (DPBS; Welgene), samples were treated for 1 h with 10% goat serum in DPBS to prevent nonspecific binding. Serum-treated cells were incubated overnight at 4°C with primary antibodies. The primary antibodies used were as follows: OCT4 (Santa Cruz Biotechnology, CA, USA; 1:200), SOX2 (Millipore; 1:200), NANOG (Santa Cruz Biotechnology; 1:200), SSEA1 (Millipore; 1:200), SSEA4 (Millipore; 1:200), Neurofilament (Millipore; 1:100), Vimentin (Millipore; 1:100), and Cytokeratin 17 (Millipore; 1:100). When we used the antibodies for intracellular proteins such as OCT4, SOX2, and NANOG, fixed cells were treated for 5 min with 0.2% Triton-X100 (Sigma-Aldrich, MO, USA) before serum blocking. After incubation with the primary antibody, the cells were treated for 3 h at room temperature with Alexa Fluor-conjugated secondary antibodies. Nuclei were stained with Hoescht 33342 (Molecular Probes). Images of stained cells were captured using a LSM 700 Laser Scanning Microscope (Carl Zeiss, Germany) and processed with the ZEN 2012 Light Edition program (Carl Zeiss).

For analysis of protein expression levels, 20 μg of total protein lysate per sample were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane, and probed with antibodies against NMNAT1 (1:1000, SantraCruz Biotechnology, Dallas, TX), NMNAT2 (1:1000, Novus Biologicals, Littleton, CO), NMNAT3 (1:1000, Abcam, Cambridge, MA), Neurofilament (1:1000, Millipore, Temecula, CA), MAP2 (1/2000, Millipore, Temecula, CA) and GAPDH (1:5000, Millipore, Temecula, CA). Western blot analysis was performed with infrared dye conjugated secondary antibodies, IR700 and IR800 (1:10000, LI-COR Biosciences, Lincoln, NE). Blots were imaged and processed on an Odyssey^® Infrared Imaging System. Densitometry analysis was performed on all the blots using ImageJ software (NIH). Data are represented as means ± SEM.