Anti cxcr4 antibody

For characterization of BMSCs, BMSCs were analysed by fluorescence‐activated cell sorting (FACS). Cells in the fourth passage were incubated in antimouse/rat CD29 FITC (1:200, eBioscience), anti‐rat CD44H PE (1:300, eBioscience), anti‐rat CD45 APC (1:400, eBioscience), antimouse/rat CD90.1 PerCP‐cyanine5.5 (1:400, eBioscience) or CD34 antibody (ICO115) FITC (1:10 dilution, Santa Cruz) at concentrations specified by the manufacturer. Corresponding isotype identical antibodies served as controls, and the concentrations of the isotype identical antibodies were the same as the labelled antibodies.
Regarding CXCR4 expression, cells were stained with anti‐CXCR4 antibody (1:50, Abcam) or IgG isotype control, and the secondary antibody was F (ab’) 2 donkey anti‐rabbit IgG PE (1:50, eBioscience). To study the effects of simvastatin on the surface expression of CXCR4, 1 μmol/l simvastatin was added into the culture medium 48 h before harvest. To determine the effect of miR‐9, cells were harvested 48 h after transient transfection.

CXCR4, p‐AKT and AKT expression was analysed by Western blot. A total of 30 μg protein extract was loaded onto a polyacrylamide gel (10%) used for electrophoresis. Then, the protein was transferred onto a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated in TBST containing 5% non‐fat dry milk for 1 h at room temperature. The membranes were then incubated with anti‐CXCR4 antibody (1:1000, Abcam), p‐AKT1/2/3 antibody (Ser 473) (1:1000, Santa Cruz), AKT1/2/3 antibody (H‐136) (1:1000, Santa Cruz) or rabbit anti‐GAPDH (1:1000, Good Here) at 4°C overnight followed by peroxidase‐conjugated AffiniPure goat anti‐rabbit IgG (H+L) (1:5000) for 1 h at room temperature. Immunoreactivity was revealed by chemiluminescence. The relative integrated density values were measured based on the GAPDH protein expression levels as the control.
Different concentrations of simvastatin were added into the culture medium at different concentrations 48 h before the Western blot analysis as follows: 0. 1 μmol/l, 0.33 μmol/l, or 1 μmol/l. To examine the effect of p‐AKT on CXCR4 expression, the BMSCs were pre‐treated with or without the PI3K inhibitor LY294002 (30 μmol/l) for 2 h prior to treatment with simvastatin (1 μmol/l) for 48 h.

Formalin-fixed paraffin-embedded (FFPE) tumor tissue sections were deparaffinized in xylene and then subjected to a gradient of alcohol, followed by retrieval in IHC-TekTM Epitope Retrieval Steamer with IHC-TekTM Epitope Retrieval Solution (IHC World) following the instruction of the manufacturer. To block endogenous peroxidase activity, the sections were incubated in 3% H₂O₂ for 10 min, followed by incubation in blocking buffer containing 2% horse serum, 1% BSA, 0.1% Triton X-100, 0.05% Tween 20 in PBS at room temperature for 30 min. Then sections were incubated with anti-CXCR4 antibody (1:400, Abcam) at 4 °C overnight. After washing, sections were incubated with goat anti-rabbit IgG-HRP (1:500, Abcam) for 1 h and visualized with DAB. The slides were analyzed with Keyence Microscope BZ-X810.

Human dental pulp stem cells were seeded on coverslips overnight and fixed in 4% paraformaldehyde solution in PBS for 15 min at 4 °C. After washing with PBS three times, cells were permeated with 0.1% Triton-100 for 10 min and blocked by 5% bovine serum albumin (BSA) for 30 min at room temperature. Cells were then incubated at 4 °C with diluted anti-CXCR4 antibody (Abcam, 1:100) overnight. Fluorescein isothiocyanate (FITC) labeled donkey anti-rabbit secondary antibodies were added onto cells for 1 h at 4 °C after washing. Pictures were taken with Olympus FV1000 confocal microscope and acquired using the FV10-ASW3.1 Viewer software. After seeding for 12 hours and culture in α-MEM with 10% FBS and 50 ng/ml SDF-1α (Gibco, USA) for 2 hours, primary antibodies against phospho-FAK (Y397, Abcam) and β-catenin (Cell Signaling Technology) were used as described above. Dilutions without primary antibodies served as negative control.