Ab49874

To assess the transplantation site that forms nodular structures, the fixed proximal nerve portions were cut into 30 μm longitudinal frozen sections. To evaluate CNS cells in the transplant environment and the survival of the engrafted neurons, we used the following stains: Alexa Fluor 488 conjugated anti-Tuj1 antibody (801203, 1:400, Biolegend), Cy3 conjugated anti-GFAP antibody (ab49874, 1:400, Abcam, Cambridge, England), anti-MBP antibody (ab40390, 1:200, Abcam), anti-ChAT antibody (AB144P, 1:50 Sigma-Aldrich), and Hoechst (33342, 1:1000, Dojindo) for nuclear staining. Donkey Anti-Rabbit IgG H & L (Alexa Fluor 647) (ab 150075, 1:200, Abcam) for anti-MBP antibody and Donkey Anti-Goat IgG H & L (Alexa Fluor 594) pre-absorbed (ab 150136, 1:200, Abcam) for anti-ChAT antibody were used as secondary antibodies. We also stained the naive spine and common peroneal nerve with the same antibodies for comparison. We observed these sections using a confocal laser microscopy system (A1Rsi and Ti-E, Nikon, Tokyo, Japan).

Brain tissue slides were blocked in 1% bovine serum albumin/phosphate-buffered saline for 1 h at RT and incubated with anticluster of differentiation 31 (CD31) (1 : 200, Cat. No. ab182981, Abcam), anti-ionized calcium binding adaptor molecule 1 (1 : 250, Cat. No. ab178847, Abcam), antiglial fibrillary acidic protein (1 : 400, Cat. No. ab49874, Abcam), and antineuronal nuclear protein (1 : 250, Cat. No. ab177487, Abcam) at 4°C for overnight, followed by incubation with corresponding secondary antibodies at RT for 1 h. The experiment was repeated three times with consecutive brain slices from each group, and the slides were observed by fluorescence microscopy in a blinded fashion.