Cyanine3 (cy3)

The fixed NSCs and a 4-μm-thick longitudinal slice of spinal cord (centered on the epicenter of the injured lesion) were prepared for immunofluorescence staining as described in our previous study [42 (link)]. The primary antibodies were used as follows: mouse anti-2′3′ cyclic nucleotide 3' phosphodiesterase (Cnpase,1:200; Abcam, UK) and rabbit anti-myelin basic protein(MBP, 1:300; Abcam, UK) for oligodendrocytes, rabbit anti-glial fibrillary acidic protein (GFAP) for astroglia (1:1000; Abcam, UK), mouse anti-nestin for NSCs (1:1000; Abcam, UK), rabbit anti-neuron-specific class III beta-tubulin (Tuj1) for neuron (1:1000; Abcam, UK),rabbit anti- SRY-Box transcription factor 10 (Sox 10) for NSCs (1:100; Abcam, UK), rabbit anti- p75 neurotrophin receptor (p 75) for NSCs (1:100, Abcam, UK), and rabbit anti-BMP2 (1:500; Affinity, USA). The secondary antibodies used were Cy3 (red, 1:50; Elabscience, China) and Alexa Fluor 488 (green, 1:50; Elabscience, 288 China). The images were observed and photographed by using a DM-6B fluorescence microscope (Leica, Germany). The percentage of positive areas was calculated using ImageJ. For cell counting, random fields containing a total of 500 cells were randomly selected. The number of positive cells was blindly quantitated in two different individuals.

Spinal cord tissues were obtained from the rats 4-weeks post-injury and fixed in 4% paraformaldehyde. A 4-μm-thick longitudinal slice was obtained using a Leica RM2135 electric slicer. The following primary antibodies were used in the overnight incubation at 4°C: rabbit anti-Map-2 for neurons (1:500; Abcam, United Kingdom), mouse anti-glial fibrillary acidic protein (GFAP) for astroglia (1:1,000; Abcam, United Kingdom), and myelin basic protein (MBP) for oligodendrocytes (1:1,000; Abcam, United Kingdom) followed by incubation with primary antibodies for 1 h at room temperature: Alexa Fluor 488 (green, 1:50; Elabscience, China) and Cy3 (red, 1:50; Elabscience, China). The sections were observed and photographed using a DM-6B fluorescence microscope (Leica, Germany) connected to a computer screen. The percentage of positive areas was calculated using Image J.