Anti cxcr4 rabbit polyclonal antibody

For immunohistochemistry, the following antibodies were used: anti-CD138 mouse (B-A38) monoclonal antibody (Cell Marque, CA, USA) and anti-CXCR4 rabbit polyclonal antibody (Abcam). After deparaffinization and rehydration, the slides were placed in a pressure cooker in 0.01 M citrate buffer (pH 6.0) and were heated for 7 min. Incubation with the different antibodies was carried out overnight at 4°C. Detection was performed with DAKO en vision system according to the manufacturer′s protocol. For double immunofluorescence, primary antibodies were detected by incubation with the following secondary antibodies: donkey anti-rabbit conjugated with Dylight 488 (Jackson ImmunoResearch, Suffolk, UK) and donkey anti-mouse conjugated with Cy5 (Jackson ImmunoResearch). After incubation of slides with conjugated secondary antibody for 30 min, slides were counterstained and mounted with mounting medium (Vectashield, Vector laboratories, Burlingame, CA, USA).

Gemcitabine (GEM) was purchased from Eli Lilly (Indianapolis, IN, USA). AMD3100, MTT, Hoechst 33342, PD98059 (an ERK inhibitor), PF573228 (a FAK inhibitor), SB20938 (a P38 inhibitor) were all supplied by Sigma-Aldrich (St. Louis, MO, USA). Recombinant human SDF-1α (rhSDF-1α), the anti-IL-6 mouse monoclonal antibody (mAb) and the IL-6 ELISA kit were purchased from R&D Systems (Minneapolis, MN, USA). The anti-αSMA mouse mAb was from Dako. The anti-vimentin and anti-SDF-1α mouse mAbs, the anti-CXCR4 rabbit polyclonal antibody and the SDF-1α neutralizing antibody (SDF-1α Ab) were from Abcam. Rabbit mAbs against pAKT (Ser473), pP38 (Thr180/Tyr182), ERK1/2, pERK1/2 (Thr202/Tyr204) and pFAK (Tyr397) were from Cell Signaling Technologies (Beverly, MA, USA). The mouse mAb against FAK, rabbit mAbs against AKT and and β-actin, the rabbit polyclonal antibody against P38, and the secondary horseradish peroxidase-conjugated anti-rabbit and anti-mouse antibodies were all purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

The streptavidin-peroxidase-biotin immunohistochemical staining method was used to study the expression of SDF-1α and CXCR4 in tissue microarray samples. Briefly, paraffin-embedded specimens were cut into 4-μm sections and were baked at 60 °C for 1 h. Endogenous peroxidase activity was quenched by incubation in 3 % hydrogen peroxide/methanol buffer for 30 min. The sections were incubated in rabbit anti-SDF-1α polyclonal antibody (1:200; Abcam, UK) or rabbit anti-CXCR4 polyclonal antibody (1:400; Abcam, UK) overnight at 4 °C in humidified chambers. The following day, the sections were washed three times in PBS and were incubated in a peroxidase-conjugated goat anti-rabbit IgG antibody, which came from the streptavidin-peroxidase-biotin reagent kit, for 30 min at 37 °C. After being washed in PBS, the tissue sections were stained with diaminobenzidine, counterstained with hematoxylin, and then examined under a light microscope. As negative controls, tissue sections were processed as described above, except that they were incubated overnight at 4 °C in blocking solution with PBS.