Anti human cxcl12 antibody

Recombinant Human CXCL12 and anti-human CXCL12 antibody were purchased by R&D system Inc. (Minneapolis, MN, USA). LY294002 (PI3K inhibitor) was ordered from Cell Signaling Technology (Beverly, MA, USA). The monoclonal antibodies (mAbs) included PTEN antibody, phospho-PTEN (ser380) antibody, Akt antibody, phospho-Akt (ser473), PI3K p85 antibody, phospho-PI3K p85 (Tyr 458) /p55 (Tyr199) antibody were provided by Cell Signaling Technology.

Commercial MM BM tissue microarray containing four cases of myeloma and two normal BM tissue samples was used (US Biomax). Formalin-fixed, paraffin-embedded tissue samples were initially dewaxed, rehydrated, treated with EDTA buffer and blocked with CAS blocking reagent (Zymed Laboratories) for 30 minutes atroom temperature. Samples were then incubated overnight at 4°C in a humidified chamber with anti-human CXCL12 antibody (R&D Systems), and diluted to a final concentration of 10 μg/ml. Next, the sections were incubated with secondary anti-mouse horseradish peroxidase-conjugated antibody (DakoCytomation) for 30 minutes at room temperature. 3-amino-9-ethylcarbazole (AEC) was used for color development, and sections were counterstained with hematoxylin.