Anti cxcr4 ab 2074

Whole cell lysates were obtained by harvesting cells with Laemmli buffer, containing 100 mM Tris (pH 6.8), 20% glycerol, 4% SDS. Protein concentration was determined by BCA protein assay kit (Pierce) following the manufacturer's instructions. Then, DTT (200 mM) was added and lysates were boiled for 5 min. Proteins were separated in SDS polyacrylamide gels and transferred to nitrocellulose by standard procedures. The following antibodies were used to detect the proteins of interest: anti-CXCR4 (Ab-2074; Abcam, UK); anti-ZEB1 (H-102; Santa Cruz Biotechnology, Heidelberg, Germany); anti- human p53 (Ab-6, DO-1 Oncogene Research Products); anti-murine p53 (Ab-1; Oncogene Research Products, Boston, MA, USA); anti-actin (AC15; Sigma).

Paraffin-embedded EWS specimens were incorporated in tissue microarrays (TMAs) and processed for immunohistochemistry (IHC) using an avidin–biotin–peroxidase method (Vector Laboratories, Inc., Burlingame, CA, USA). An overnight incubation with the following primary antibodies was performed: anti-CD164 (sc-271179, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:50, anti-CXCR4 (ab2074, Abcam, Cambridge, UK) diluted 1:50, anti-IGF2BP3 (sc-47893, Santa Cruz Biotechnology) diluted 1:50. Samples were classified as follows: negative, when no staining was observed; positive when weak, moderate, or strong staining was observed.