Anti cxcr4

De-identified human triple negative breast cancer specimens were obtained from the Tissue Procurement Core of Siteman Cancer Center under the approval from Human Research Protection Office of Washington University. The Tissue Procurement Core consented the patients for using their biospecimens for research projects. In cased where consent was not obtainable, the Tissue Procurement Core has an Institutional Review Board issued waiver of consent, under which tumor tissues may be used anonymized biospecimens for non-genomic based studies. Paraffin embedded sections were deparaffinized in xylenes and rehydrated through a series of graded alcohols. Tissues were processed for antigen retrieval by boiling in citrate buffer (pH 6.0 containing 0.1% Tween). They were blocked in 10% donkey serum for 2 h to prevent nonspecific binding. The sections were then incubated overnight at 4 °C in primary antibody (anti-CXCR4, Novus Biologicals 1:400) or control IgG (anti-rabbit IgG, Novus Biologicals, 1:400). Anti-rabbit secondary antibody was applied (Jackson Laboratories) for 1 h at room temperature, and sections were washed in PBS, mounted in DAPI mounting medium (Vector Laboratories), and imaged using a Zeiss Confocal microscope system.

Paraffin embedded tumor sections were deparaffinized in xylenes and rehydrated through a series of graded alcohols. After quenching endogenous peroxidase activity in 3% hydrogen peroxide, antigen retrieval was performed by boiling the slides in citrate buffer (pH 6.0 containing 0.1% Tween). After incubation in blocking serum for 1 h to prevent nonspecific binding (Vectastain; Vector Laboratories), the sections were further incubated overnight at 4°C with primary antibody (anti-CXCR4, Novus Biologicals 1:2000) or control IgG (anti-rabbit IgG, Novus Biologicals 1:2000). After applying secondary antibody (Vector Laboratories), the addition of diaminobenzidine develop a brown color. The sections were counterstained in hematoxylin, and digital images of the stained sections were collected using a Zeiss Light Microscope system.