Rabbit anti p75ntr

To examine if TBI up-regulates p75NTR expression in the circulation, we used flow cytometry with leukocytes isolated from C57Bl/6 mice at 1 and 6 weeks after TBI with or without EVT901. The leukocytes were isolated as described earlier. The isolated cells were incubated with AARD according to the manufacturer’s protocol. For intracellular staining, the isolated leukocytes were fixed with IC fixation buffer (eBioscience) and treated with permeabilization buffer (eBioscience). The fixed leukocytes were stained with rabbit anti-p75NTR (Abcam) for 30 min at 4 °C and then stained with anti-rabbit-PE Ab (Invitrogen) for 30 min at 4 °C. The p75NTR-expressing cells were identified by flow cytometry (LSRII, BD Bioscience). Non-specific signal was determined using isotype control Ab. A total of 34 C57Bl/6 WT mice were used: n = 4–5 per TBI group (vehicle vs EVT901) and n = 3–5 for sham groups (vehicle vs EVT901).

hE-SCs/P0, hE-SCs/P2,and hE-FLCs/P2 were trypsinized and replated on cover slides for immunostaining. The cells were fixed with 4% paraformaldehyde (Sigma) in PBS for 15 min. The fixed cells were washed three times with PBS for 5 min each, and then, the cells were blocked with 10% bovine serum albumin (Sigma, USA) in PBS for 30 min at room temperature, followed by three washes with PBS. The cells were then incubated with rabbit anti-glial fibrillary acidic protein (GFAP; 1:500; Abcam, Cambridge, UK), rabbit anti-P75NTR (1:500 diluted in PBS; Abcam, Cambridge, UK), rabbit anti-S100 (1:500; Dako, Glostrup, Denmark), rabbit anti-Sox10 (1:500 diluted in PBS; Abcam, Cambridge, UK), and rabbit anti-Krox10 (1:500 diluted in PBS; Abcam, Cambridge, UK) overnight at 4 °C. After being washed with PBS, the slides were treated with Alexa Fluor 488 goat anti-rabbit IgG (1:500; Invitrogen, USA) at 37 °C for 60 min. After they were washed with PBS, the cells were incubated with 1 M Hoechst 33343 (Sigma, USA) for 10 s. Labeled cells were examined with fluorescence microscopy (Olympus, Japan), and the images were recorded and processed with Image-Pro Plus (Media Cybernetics, USA).