All formalin-fixed paraffin-embedded tissue samples were provided from the Institute of Pathology, University Hospital Duesseldorf. The construction of the tissue microarrays, immunohistochemistry and analysis of protein expression using the immunoreactivity score (IRS) reported by Remmele (Remmele et al, 1986 (link)) were performed as described previously (Werner et al, 2016 (link)). The staining intensity of the different samples was reviewed by two independent investigators (TW and CF) in a blinded manner.
For immunohistochemical staining the following primary antibodies were used: mouse monoclonal anti-CXCR4 (1 : 100 dilution; Abcam, Cambridge, UK), rabbit polyclonal anti-CXCR7 (1 : 200 dilution; GeneTex, Irvine, CA, USA).
Isotype controls were performed using mouse IgG1k (MOPC-21; 1 : 50 dilution; Abcam) or 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 according to the REporting recommendations for tumour MARKer prognostic studies (REMARK) (McShane et al, 2005 (link)).
For immunohistochemical staining the following primary antibodies were used: mouse monoclonal anti-CXCR4 (1 : 100 dilution; Abcam, Cambridge, UK), rabbit polyclonal anti-CXCR7 (1 : 200 dilution; GeneTex, Irvine, CA, USA).
Isotype controls were performed using mouse IgG1k (MOPC-21; 1 : 50 dilution; Abcam) or 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 according to the REporting recommendations for tumour MARKer prognostic studies (REMARK) (McShane et al, 2005 (link)).
