Anti cxcl12

The human PCa cell lines CFPAC-1, BxPC-3, MiaPaCa-2, AsPC-1, SW1990, Panc-1, and the rat Schwann cells RSC96 were purchased from the Chinese Academy of Sciences Cell Bank of Type Culture Collection (CBTCCCAS). According to the instructions, all cell lines were cultured in the proper medium (HyClone, Logan, USA) supplemented with 10% fetal bovine serum (FBS), 100 μg/ml ampicillin and 100 μg/ml streptomycin. The cultures were incubated at 37°C in a humidified atmosphere containing 5% CO₂.
Recombinant human CXCL12 was purchased from PeproTech (Rocky Hill, USA). The pharmacological reagent AMD3100 was purchased from Sigma (St. Louis, MO, USA). Antibodies were purchased from the following sources: anti-CXCR4, anti-CXCL12, anti-NGF, anti-β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MMP-2, anti-uPA (Bioworld, Minneapolis, MN, USA), anti-S100 and anti-NF200 (Abcam, USA).

Mice were deeply anesthetized and perfused transcardially with 25 mL of ice‐cold phosphate‐buffered saline (PBS), followed by 20 mL of 4% paraformaldehyde (PFA). Brains were post‐fixed in 4% PFA for 24 h and dehydrated in serial 15% and 30% sucrose solutions at 4 °C. Next, the brain samples were sectioned into 25 µm thick coronal slices. The sections were stored in cryoprotectant (40% PBS, 30% glycerol, 30% ethylene glycol) and kept at −20 °C until immunostaining. Brain sections were washed twice with PBS, followed by permeabilization in 0.5% Triton X‐100 at room temperature. Next, brain sections were blocked with 5% normal donkey serum in PBS for 1 h at room temperature and incubated overnight at 4 °C with the following primary antibodies: anti‐CD31 (Santa Cruz, sc18916, 1:50), anti‐IBA1 (Abcam, ab5076, 1:250), anti‐CXCL12 (Santa Cruz, sc74271, 1:50). The sections were then incubated in the dark with donkey secondary antibody conjugated with Alexa Fluor 555 or 647 (Invitrogen, 1:500) at room temperature for 1 h. After washing with PBS for three times, the sections were mounted on glass slides with mount‐G containing DAPI (Yeasen Biotech). Sections were observed and analyzed with a Leica TCS SP8 confocal microscope (Leica Microsystems). Images were adjusted for brightness and contrast using Fiji 2.1.0/1.53c. All confocal images were represented as maximum intensity projections.