Rabbit anti p2x4rs

On the 14th day after surgery, the animals were deeply anesthetized with penthiobarbital sodium and transcardially perfused from the ascending aorta with normal saline and 4% paraformaldehyde (PFA) in 0.1 mol/L phosphate buffer (pH 7.4), respectively. After L_4–5 spinal cords were removed, post fixed in same fixative for 3 hr, and dehydrated by 30% sucrose in phosphate buffer over two nights. After being cut into 25‐μm thick sections using a cryostat, the transverse spinal sections were processed for immunofluorescence. All the blocked sections were blocked with 5% goat serum in 0.3% Triton X‐100 for 1 hr at room temperature (RT) and incubated with mouse anti‐iba1 (1:500, Abcam) and rabbit anti‐P2X4Rs (1:200, Alomone Labs) over night at 4°C. After three rinses in PBS, all the sections were treated by a mixture of Alexa 647 donkey anti‐mouse IgG and Alexa 488 donkey anti‐rabbit IgG (1:200, Thermo Fisher) for 2 hr at RT, and thereafter, the preparations were washed in PBS. Olympus IX73 (Olympus Optical, Olympus Co. Ltd., Tokyo, Japan) fluorescence microscope was used to analyzed the stained sections, and images were captured with a CCD spot camera. The primary antibodies were omitted during staining when serving as a negative control, and further examined by Western blot.

Ipsilateral spinal cord tissues were collected and homogenized in 1,000 µl ice‐cold cytoplasmic lysis of RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, CN) and phosphatase inhibitor cocktail (CWBIO, Beijing, CN). Then, the homogenate sample was centrifuged at 12,000 rpm for 15 min at 4°C and collected the supernatant containing membrane protein samples. Protein samples were dissolved in loading buffer and denatured at 99°C for 5 min. 30–60 μg protein was subjected to 8%–12% SDS polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membranes (PVDF, Millipore, Bedford, MA, USA). The PVDF membranes were blocked in 5% skim milk for 2 hr and then incubated overnight at 4°C with the following primary antibodies (rabbit anti‐P2X4Rs, 1:200, Alomone Labs; rabbit anti‐BDNF, 1:1,000, Abcam; rabbit anti‐p38, Abclonal; rabbit anti‐p‐p38, Abclonal and rabbit anti‐GAPDH, 1:500, Goodhere Biotechnology Co.). The proteins were detected with a HRP‐conjugated goat anti‐rabbit secondary antibody IgG (1:5,000, Beijing Zhongshan Biotech Co.). Specific bands of target proteins were visualized using the chemiluminescence reagents provided with the ECL kit (Solarbio, Beijing, CN) and exposed to film. The band densities were determined using Image J Software and normalized to each internal control.