Ab16829

Sections were obtained as mentioned above, and the slides were incubated overnight. The slides were washed for 15 min in 0.1% Triton X-100 diluted with PBS and blocked with 5% bovine serum albumin (ZSGB-BIO, Beijing, China) for 30 min. The slides were incubated in the following antibodies overnight at 4°C: CD31 anti-collagen I (1:2000, ab182981, Abcam, Cambridge, United Kingdom), endomucin anti-collagen I (1:50, sc-65495, Santa Cruz Biotechnology, Santa Cruz, CA, USA), PDGF-BB anti-collagen I (1:100, ab16829, Abcam, Cambridge, United Kingdom), VEGFA anti-collagen I (1:100, ab46154, Abcam, Cambridge, United Kingdom), HMGB1 anti-collagen I (1:100, ab79823, Abcam, Cambridge, United Kingdom). The slides were washed with PBS AND incubated with a fluorescent secondary antibody (AlexaFluor®488) for 50 min at 37°C. DAB was used to stain the nuclei. The slides were observed under a fluorescence microscope (Motic, Wetzlar, Germany). ImageJ software (National Institutes of Health, Bethesda, MD, USA) was used to images the results at 200× magnification.

PFA-fixed L4 spinal cord segments were embedded in O.C.T. compound (Tissue-Tek, Sakura, USA), and 10-μm serial transverse sections were sliced using in a cryostat. The sections were blocked with 5% normal goat serum for 2 h and incubated overnight at 4 °C in a humidified chamber with the following primary antibodies: rabbit polyclonal anti-PDGF BB (1:100) (ab16829, Abcam), mouse monoclonal anti-NeuN (1:400) (ab104224, Abcam), mouse monoclonal anti-GFAP (1:50) (MAB3402x, Millipore), mouse monoclonal anti-OX-42 (1:100) (MCA275G, AbDsreotec), rabbit polyclonal anti-RFP (1:400) (r10367, Abcam), rabbit polyclonal anti-SP (1:100) (AB1566, Chemicon) and rabbit polyclonal anti-CGRP (1:200) (ab22560, Abcam). The primary antibodies were dissolved in 2% normal goat serum. The sections were washed 3 times with PBS for 5 min each and incubated with the appropriate secondary antibodies: goat anti-rabbit (1:400) (A-24921, Invitrogen) and goat anti-mouse (1:400) (A-24920, Invitrogen) for 2 h at 37 °C. The sections were washed with PBS 3 times for 5 min each, stained with DAPI and mounted. To confirm the specificity of the PDGF labeling, the synthetic immunogen (HangZhou HuaAn Biotechnology Co. Ltd) was pre-incubated with the PDGF-BB antibody. Then, sections in this experimental control were performed by the same process as described above (see Supplementary Fig. S1).

The induced membrane proteins were collected by lysis in RIPA buffer, a BCA protein assay was used to measure the protein content. After centrifugation for 10 min, the extracted protein was put in boiling water for 10 min. The proteins were isolated by 10% polyacrylamide gel electrophoresis at 120 mV, and transferred to PVDF membranes at 200 mA for approximately 1h. Subsequently, the membranes were blocked using 5% non-fat dried milk and 4% BSA for 2 h at 25°C, followed by incubation with the following primary antibodies: anti-GAPDH (1:1000, Shanghai Kangcheng Co., China), anti- HMGB1 (1:1000, ab79823, Abcam, Cambridge, United Kingdom), anti-VEGFA (1:1000, ab46154, Abcam, Cambridge, United Kingdom), anti-CD31 (1:1000, ab182981, Abcam, Cambridge, United Kingdom), and anti-PDGF-BB (1:1000, ab16829, Abcam, Cambridge, United Kingdom), anti-Endomucin (1:1000, sc-65495, Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, the membranes were incubated with the appropriate horseradish peroxidase (HRP)-conjugated secondary antibody (1:10,000, ZB-2301, Wuhan Bode Biological Engineering Co, Ltd, Wuhan, China) for 1h on a shaker at 37°C. Finally, after ECL chemiluminescence, development, and fixation, the film was scanned. Sensiansys software in a JS-680A automatic gel imaging analysis system was used for the grayscale analysis of the target bands.