Rabbit anti p75

Monolayer-cultured rBM-NCPs were fixed with 4% (wt/vol) paraformaldehyde, blocked, and then incubated with primary antibodies, including rabbit-anti-CD133 (1:200, Abcam), rabbit-anti-p75 (1:1000, Cell Signaling), rabbit-anti-nestin (1:50, Sigma), mouse-anti-vimentin (1:25, Sigma), mouse-anti-CD29 (1:100, Sigma), and rabbit-anti-Ki67 (1:200, Sigma), overnight at 4 °C, followed by reaction with FITC-anti-rabbit-IgM (Sigma), Cy3-anti-rabbit-IgM (Santa Cruz), TRITC-anti-mouse-IgM (Santa Cruz) or FITC-anti-mouse-IgM (Sigma), and Hoechst 33342 (Sigma) counterstaining. The cell samples were observed under a confocal laser scanning microscope (TCS SP2, Leica Microsystems, Germany).
Induced Schwann cells were subjected to immunofluorescent staining with mouse anti-S100β (1:250, Sigma), rabbit-anti-glial fibrillary acidic protein (anti-GFAP, 1:200, DakoCytomation), and rabbit-anti-p75 (1:1000, Cell Signaling Technology) respectively, followed by the same reaction with the second antibody and Hoechst 33342 counterstaining.

Cultivated cells were fixed for 20 min using 4% paraformaldehyde (PFA), washed and permeabilized in PBS with 0.02% TritonX-100 (Sigma Aldrich) and supplemented with 5% goat serum for 30 min. The applied primary antibodies were diluted in PBS as followed: rabbit anti-Nestin 1:200 (Millipore), mouse anti-S100B 1:500 (Sigma Aldrich), rabbit anti-Slug 1:100 (Cell-Signaling Technology), rabbit anti-p75 1:500 (Cell-Signaling Technology), mouse anti-β-III-tubulin 1:100 (Promega), rabbit anti-neurofilament-L 1:50 (Cell-Signaling Technology), anti-vGlut (Millipore) and anti-Synaptophysin (Millipore). They were applied for 1 h (cells) at room temperature. After three washing steps, secondary fluorochrome-conjugated antibodies (Alexa 555 anti-mouse or Alexa 488 anti-rabbit, Invitrogen, Life Technologies GmbH) were applied for 1 h at RT with a dilution ratio of 1:300. Nuclear staining was realized by incubation with 4,6-Diamidin-2-phenylindol (DAPI) (1 μg/mL, Applichem) in PBS for 15 min at RT. Finally, the samples were mounted with Mowiol (self-made). Imaging was performed using a confocal laser scanning microscope (CLSM 780, Carl Zeiss) and image processing was executed with ImageJ and CorelDRAW [48 (link)] (open source and Corel Corporation).

The following antibodies were used as primary antibodies for immunocytostaining and for immunohistochemistry: rabbit anti‐s100b (dilution = 1:1,000) (Dako, Ely, UK), rabbit anti‐p75 (1:500) (Cell Signaling Technology, Danvers, MA, USA), rabbit anti‐GAP43 (1:200) (Cell Signaling Technology), rabbit anti‐glial fibrillary acidic protein (GFAP) (1:200) (Abcam, Cambridge, UK), rabbit anti‐NG2 (1:1,000) (Millipore, Cambridge, UK), mouse anti‐myelin basic protein (MBP) (1:100) (Chemicon, Chandlers Ford, UK), goat anti‐protein zero (P0) (1:500) (Abcam), rabbit anti‐Tuj‐1 (1:100) (Chemicon), mouse anti‐neurofilament (NF) (1:1,000) (Sigma), rabbit anti‐PGP9.5 (1:500) (Abcam), mouse anti‐fibronectin (Millipore), mouse anti‐Nanog (1:200) (ReproCELL, Yokohama, Japan), mouse anti‐MAP‐2 (1:200) (Abcam), and rabbit anti‐NeuN (1:500) (Abcam) antibodies. As secondary antibodies, Alexa Fluor 546‐conjugated anti‐rabbit and anti‐mouse antibodies and Alexa Fluor 670‐conjugated anti‐rabbit and anti‐mouse antibodies (1:1,000) (Life technologies, Carlsbad, CA, USA) were used.