Sdf 1 antibody

For immunohistochemically staining, sections were incubated by primary antibody RUNX2 (Abcam, ab76956) overnight at 4°C after standard protocol. Horseradish peroxidase-streptavidin detection system was used to detect immunoreactivity and counterstaining with hematoxylin. Immunofluorescent staining was performed using a standard protocol, we incubated sections with primary antibodies: SDF-1 antibody (Santa Cruz, sc-74271), osterix (Santa Cruz, sc-393060), p-ERK (Affinity Bioscience, AF1015), CD31 (Abcam, ab222783), and Endomucin (Emcn, Santa Cruz, sc-65495) overnight at 4°C. A second antibody conjugated with fluorescence was added to the sample sections and incubated for 1 h at room temperature (RT).

Leukemia cell line Molm13 and stromal cell line M210B4, commonly used model systems for leukemia cell-marrow interactions [22 (link)–24 (link)] (American Type Culture Collection, Manassas, VA, USA), were cultured at 37 °C in 5 % CO₂ in a humidified incubator. Both cell lines were maintained in RPMI 1640 medium supplemented with 10 % (v/v) fetal bovine serum (FBS, Invitrogen). AMD3100, a widely used drug that can selectively antagonize the binding of SDF-1 to CXCR4 and preferentially mobilize leukemic blasts into the peripheral circulation, was chosen to treat leukemia cells. Polyclonal goat anti-VCAM-1 antibodies (Santa Cruz) were used in combination with donkey anti-goat (Invitrogen) to mark VCAM-1 protein on leukemia cells. The SDF-1 protein expressed by stromal cells was stained with a rabbit polyclonal SDF-1 antibody (Santa Cruz) and goat anti-rabbit IgG-CFL 488 secondary antibody (Santa Cruz). The nucleus was visualized with DAPI.

Polyclonal goat anti-VCAM-1 antibodies (Santa Cruz) were used in combination with donkey anti-goat (Invitrogen) to mark VCAM-1 protein on leukemia cells. The SDF1 proteins expressed by stromal cells were stained with a rabbit polyclonal SDF1 antibody (Santa Cruz) and goat anti-rabbit IgG-CFL 488 secondary antibody (Santa Cruz). The nucleus was visualized with DAPI.
Cells were washed twice with 1 × PBS and fixed in 3.7 % formaldehyde for 10 min at room temperature. The cells were then washed three times and permeabilized with 0.5 % Triton X-100 in PBS. After 5 min, cells were washed again and blocked with 5 % goat serum in PBS for 20–30 min. Cells were incubated with antibody for 1 h at 37 °C, washed three times with PBS, and incubated for 45 min at 37 °C with secondary antibody. Cell nucleuses were stained with DAPI for 5 min at room temperature. The cells were then washed three more times and observed under a laser-scanning confocal microscope (Leica microsystem, Wetzlar, Germany).