Quantibody human angiogenesis array 1

Tissue and saliva samples from patients were used to measure the expression of ANG, angiopoietin-2 (ANG-2), bFGF, heparin-binding EGF (HB-EGF), HGF, LEP, PDGF-BB, PIGF, and VEGF by the Quantibody® Human Angiogenesis Array 1 (RayBiotech, USA) and MMP-1, MMP-2, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-1, and TIMP-2 by the Quantibody® Human MMP Array 1 (RayBiotech, USA). The detection was performed according to the manufacturer's instructions. Briefly, supernatant from centrifuged tissue samples treated with cell lysate and centrifuged saliva samples were used. The glass slide was completely air dry and blocked by a blocking solution for 1 h. The tissue sample diluent 100 µL (500 µg/mL) and 80 µL of the diluted tissue lysate or saliva samples were separately added to the slides and incubated overnight at 4˚C. The samples diluted were decanted from the wells and washed with wash buffer at room temperature with gentle rocking. The detection antibody cocktail was added to each well and incubated at room temperature, followed by a washing step. Afterward, Cy3 equivalent dye-conjugated streptavidin was added and incubated in a dark room, followed by a washing step. Fluorescence signals were obtained by an InnoScan 300 Microarray Scanner (Innopsys, France) at 532 nm wavelength and 10 µm resolution. Data were analyzed using Mapix (Innopsys, France) analysis software.

An ELISA based angiogenesis antibody array (Quantibody Human Angiogenesis Array 1, RayBiotech, Norcross, GA, USA) was used to determine the expression of angiogenic factors from CM secreted by SW620 cells transfected with miR-590-5p. The level of VEGF protein from indicated CM was determined with Human VEGFA₁₆₅ ELISA Kit (RayBiotech) according to the manufacturer's instructions.