Mab6838

Samples were fixed in 4% paraformaldehyde for 20 minutes followed by 1 hour treatment with blocking buffer (0.2% each of triton-x, BSA, gelatin and casein and 0.02% sodium azide). Cells were incubated in primary antibody and incubated overnight at 4°C. Following day, cells were washed in PBS and incubated with appropriate secondary antibodies (Alexa fluor series, Invitrogen, 1:2000) at room temperature for 1 hour. Cells were washed in PBS, incubated with DAPI (Invitrogen, 1:10,000) and mounted in Prolong Gold anti-fade reagent (Invitrogen). Images were captured on a Leica TCS SPII scanning laser confocal system. Primary antibodies used were as follows: Ki67 (1:400, #9027, Cell Signaling), sFRP2 (1:100, MAB6838, R&D Systems), biotinylated Wnt5A (500 ng/mL, BAF645, R&D Systems).

Nunc™ MaxiSorp™ ELISA plates (ebiosciences, CA) were coated with 50µl of 3µg/ml sFRP2 (#ab137560, abcam) overnight at 4°C. Plates were washed in PBS containing 0.1% Tween and blocked in ELISA diluent (00-4202-56, ebiosciences, CA) for 2 hours. Serum was diluted 1:100 before addition to the plates and incubated overnight at 4°C. Next day, the plates were washed in PBS containing 0.1% Tween20 and incubated with detection antibody (MAB6838, R&D systems, MN) for 1h at room temperature. Plates were washed and incubated with secondary antibody for 1h. After washing, 100µl TMB (00-4201-56, ebiosciences, CA) was added to the plates and incubated for 15 minutes. The reaction was stopped using 50µl of 2N H₂SO₄ and absorbance was measured at 450nm.