Mab172

Ethical approval for the use of paraffin-embedded samples from breast cancer tumours was submitted for proportionate review to the Research Ethics Committee (NHS Health Research Authority) and approved on the 14 March 2016 under the REC reference 16/YH/0117 (IRAS project ID 188839) by the Proportionate Review Sub-committee of the Yorkshire & The Humber—Leeds East. For the use of samples isolated prior to the Human Tissue Authority (HTA), approval had already been granted under the REC reference 06/Q0906/12. Informed consent was obtained from all subjects.
Immunohistochemistry of 4 μM paraffin-embedded cancer sections was carried out using the immunoperoxidase-based Vectastain ABC kit (Vector labs, Burlingame, CA, USA) as per the manufacturer’s instructions. Antigen retrieval was carried out using citrate buffer (pH 6) for 2 min and unspecific binding was blocked before anti-CXCR4 (1:40, MAB172, R&D systems, Minneapolis, MN, USA) or anti-ACKR3 (1:150, ab38089, Abcam, Cambridge, UK) antibodies were incubated with the sections for 2 h at room temperature. For anti-ACKR3 have used placenta as a positive control and non-immune rabbit serum as a negative control (Figure S4). Secondary and tertiary antibodies were added for 30 min before dispensing DAB on top of the samples (DAB Peroxidase (HRP) Substrate Kit, Vector labs, Burlingame, CA, USA) and counterstaining with haematoxylin.

The decidual tissues was fixed with 4% paraformaldehyde for 4 h and embedded with paraffin. Then paraffin-embedded decidual tissues was serially sectioned (thickness 5 μm). After deparaffinization and rehydration with xylene and gradient alcohol, antigenic recovery was performed by microwave heating in 0.01 M sodium citrate buffer (pH 6.0). H2O2 (0.3%) in phosphate-buffered saline (PBS) was used to block endogenous peroxidase activity in the sections. Then the sections were treated with protein blocking solution containing 5% bovine serum albumin to block non-specific binding and then incubated with mouse anti-human CXCL12 (10 μg/ml, MAB 350, R&D Systems, Abingdon, UK) or CXCR4 (25 μg/ml, MAB172, R&D Systems, Abingdon, UK) overnight at 4 °C. After three washes with PBS, the sections were overlaid with peroxidase-conjugated goat anti-mouse IgG (SP-9002; SZGB-BIO, Beijing, China), followed by 3,3-diaminobenzidine tetrahydrochloride to detect signal and hematoxylin as a counter-stain. Human first-trimester villi were used as positive control. The experiments were performed in five normal pregnancy tissues [25 (link), 27 –30 (link)].

Immunohistochemical staining of tumor specimens was carried out by using a human anti-CXCR4 monoclonal antibody (1 : 1000 dilution, MAB172, R&D Systems) with BOND equipment (Leica Biosystems). The analysis was performed double-blind by two pathologists, which selected five random fields for each tumor specimen under an optical microscope (magnification 200x). Gastric cancer tissue was used as an internal positive control for the expression of CXCR4.

Immunofluorescence was used to detect the expression and position of CXCR7 and CXCR4 in DECs. The isolated DECs were seeded on 6-well plates which were pre- coated with cover glasses at a density of 2 × 10⁶ cells/ml × 2 ml per well. At 80–90% confluence, the cells were fixed in 4% paraformaldehyde and treated with 0.2% Triton X-100. After blocking with goat serum for 2 h at room temperature, the cells were incubated with mouse anti-human CXCR4 (25 μg/ml, catalogue number MAB172, R&D Systems, Abingdon, UK) and rabbit anti-human CXCR7 monoclonal antibodies (20 μg/ml, catalogue number ab72100, Abcam, Cambridge, MA,). After three washes with PBS, the cells were incubated with fluoresce-in isothiocyanate (FITC) conjugated donkey anti-rabbit immunoglobulin (Ig) G (1:100, 10 μg/L) or Texas Red conjugated donkey anti-mouse IgG (1:100, 10 μg/L) (Rockland) at room temperature for 60 min in darkness and then incubated with DAPI (Santa Cruz Biotechnology) for 5 min at 37 °C. Images were captured with an Olympus fluorescence microscope (Olympus, Tokyo, Japan). The experiments were perform using three samples.

TMA sections were deparaffinized and rehydrated. Antigen retrieval was achieved in 10 mM sodium citrate buffer at pH 6.0, using microwave for 3 cycles of 10 min at 1-fold 2 min at 600 W and 2-fold 4 min at 360 W each. Slides were treated with 3% hydrogen peroxide for 10 min at 24 °C to stop endogenous peroxidase, and then blocked in biotin/avidin solution for 10 min. Immunostaining was performed by incubating with anti-CXCR4 (1:100, MAB172, R&D systems) or anti-CXCR7 (1:70, MAB42273, R&D systems) or anti CXCL12 (1:50, MAB350, R&D systems) at 4 °C overnight. Anti-mouse secondary antibodies and peroxidase-labeled streptavidin were used (Dako, Carpinteria, California, CA, USA); diaminobenzidine reagent was employed to detect signals. The staining intensity was scored as 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The percentage of cells stained was counted. The H-Score was calculated [22 (link)]. The markers’ expression was separately evaluated on epithelial ovarian cancer cells and stromal cells within each TMA core.