Mouse monoclonal anti cxcr4

Western blot analysis was performed as reported previously (24 (link)). Cells were lysed in radioimmunoprecipitation assay lysis buffer (EMD Millipore, Billerica, MA, USA). Total protein concentration was determined using a Bradford Protein Assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) (25 (link)). Extracted protein (~30 µg) was subjected to electrophoresis on a 10% SDS polyacrylamide gel, and was then transferred to a polyvinylidene difluoride membrane (EMD Millipore Corporation, Billerica, MA, USA) at 80 V for 2 h at 4°C. The membrane was blocked in skim milk for 1 h, and subsequently incubated overnight at 4°C with a mouse monoclonal anti-CXCR4 (Abcam; dilution 1:2,000; #ab58176) or a rabbit monoclonal anti-EGFR (Cell Signaling Technology, Inc., Danvers, MA, USA; dilution 1:3,000; #4405) antibody. The following day, the membrane was gently washed in Tris-buffered saline with 0.1% Tween-20 (TBST), and then incubated with goat anti-mouse (#ZB-2305) or goat anti-rabbit (#ZB-2301) horseradish peroxidase-conjugated secondary antibodies (OriGene Technologies, Inc.; dilution 1:2,000) for 2 h at room temperature. Subsequent to washing in TBST, the immunocomplexes were detected using ECL Plus reagent (Applygen Technologies, Inc., Beijing, China) (16 (link),26 (link)).

Cells were cultivated in 6-well chamber slides and fixated with ice-cold methanol. After treatment with 0.5 % Triton the cells were blocked with 20 % AB serum and incubated with their respective primary antibody and isotype control (mouse monoclonal anti-CXCR4, 1:250 dilution; Abcam, Cambridge, UK; rabbit polyclonal anti-CXCR7, 1:500 dilution; GeneTex, Irvine, CA, USA; mouse IgG1k (MOPC-21); 2 µg/ml; Abcam, Cambridge, UK; rabbit immunoglobulin fraction (Code X0903) 2 µg/ml; Dako, Glostrup, Denmark). After 45 minutes of incubation, cells were washed and incubated with anti-mouse- or anti-rabbit-Alexa Fluor® 488 (10 µg/ml; Thermo Fisher, MA, USA), respectively. Nuclear staining was carried out with DAPI (200 ng/ml). After fixating the cells with 1 % PFA, visualization was achieved using a fluorescence microscope at 400x magnification (Zeiss Axioplan 2, Carl Zeiss, Göttingen, Germany).

Cells were harvested using ice-cold RIPA buffer. Total protein concentrations were determined using a BioPhotometer® according to Bradford (Eppendorf, Hamburg, Germany). Protein separation was achieved by SDS-polyacrylamid gel electrophoresis and subsequent transfer onto a nitrocellulose membrane. Antibody-detection was performed overnight using the following primary antibodies: mouse monoclonal anti-CXCR4 (1:100 dilution; Abcam, Cambridge, UK), rabbit polyclonal anti-CXCR7 (1:200 dilution; GeneTex, Irvine, CA, USA).

Formalin-fixed paraffin-embedded tissue samples were obtained from the Institute of Pathology, University Hospital Duesseldorf. All tissue samples were newly reviewed prior to this study and the diagnosis of FTC histologically confirmed. FTCs were defined as thyroid carcinomas with follicular differentiation in the absence of papillary nuclear features, with either capsular or vascular invasion. The construction of the tissue microarray, immunohistochemistry and protein expression analyses were performed as described previously21 (link),22 (link). The expression levels of CXCR4 and CXCR7 were scored by two independent investigators (TW, CF) using the immunoreactivity score (IRS) reported by Remmele23 (link) in a blinded manner.
The immunohistochemistry was carried out using mouse monoclonal anti-CXCR4 (1:100 dilution; Abcam, Cambridge, UK) and rabbit polyclonal anti-CXCR7 (1:200 dilution; GeneTex, Irvine, CA, USA) as primary antibodies. Isotype control was performed using mouse IgG1k (MOPC-21; 1:50 dilution; Abcam, Cambridge, UK) and rabbit immunoglobulin fraction (Code X0903; 1:1000 dilution; Dako, Glostrup, Denmark). CXCR4 expressing tonsil tissue and CXCR7 expressing pancreatic adenocarcinoma served as positive controls. The prognostic power of CXCR4 and CXCR7 was assessed in accordance with the REporting recommendations for tumour MARKer prognostic studies (REMARK)24 .