Mouse anti 2 3 cyclic nucleotide 3 phosphodiesterase cnpase

The cells were lysed using RIPA lysis buffer (Beyotime, P0013B) with freshly added 1% phenylmethylsulfonyl fluoride (PMSF, Amresco, O754) solution. Protein concentration was determined using Coomassie brilliant Blue G-250. SDS-PAGE and Western blotting were carried out as reported previously (Niu et al., 2012a (link)). Proteins were transferred to polyvinylidene difluoride (PVDF) membranes and visualized by chemiluminescence (ECL Plus, GE Healthcare, Marlborough, MA, USA) after incubation with the antibodies. β-actin was used as the loading control. Quantification of band intensity was performed using ImageJ software. The primary antibodies included the following: rabbit polyclonal anti-RyR3 (1:1000, Millipore), rabbit polyclonal anti-platelet-derived growth factor receptor α (PDGFRα; 1:1000, Santa Cruz, sc-338), mouse anti-2′,3′-cyclic nucleotide-3′-phosphodiesterase (CNPase; 1:1000, Abcam, ab6319), goat anti-MBP (1:1000, Santa Cruz) and mouse anti-β-actin (1:2000, Santa Cruz, sc-47778). The secondary antibodies included the following: goat anti-mouse-HRP (1:2000, Santa Cruz, sc-2094), goat anti-rabbit-HRP (1:2000, Santa Cruz, sc-2313) and rabbit anti-goat-HRP (1:2000, Santa Cruz, sc-2020).

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.