Tumour specimens from breast cancer patients treated at the Breast Unit at Guy's Hospital London, with prospectively acquired long-term follow-up (25 years) were used in this study.
Using tissue microarrays (TMAs), of primary breast tumour specimens from a series of 252 invasive ductal and lobular carcinomas were immunolabelled for CXCR4. Polyclonal antibodies to human CXCR4 (Anti-Human CXCR4 ARP4016){CXCR4 peptide ARP 7039 N-terminal extracellular domain (1–38)} and two further anti-human CXCR4 cytoplasmic antibodies against two distinct peptides based on the membrane proximal sequence (GAKFKTSAQHALTSVSRG) and distal cytoplasmic sequence (VSTESESSSFHSS) of huCXCR4 cytoplasmic domain, were used to detect CXCR4.
The immunohistochemical detection of CXCR4 expression, was assessed by 2 independent pathologists (with consensus agreement). Both the proportion and intensity of expression was recorded for the total and subpopulations of CXCR4 recognised by ARP4016 and the two cytoplasmic antibodies, respectively.
For immunofluroscence the average fluorescence intensity/unit area of cells stained with the respective antibodies were plotted and quantified.