Invitrogen evos fl auto cell imaging system

To assess the angiogenic effects of ciPVT1 in vivo, we used a well‐established Matrigel plug assay. Six‐week‐old male BALB/c nude mice were administered a subcutaneous injection of Matrigel (BD Biosciences) supplemented with saline, VEGF, or mixed with HUVECs. After 7 days, the animals were euthanized and the Matrigel plugs were removed, weighed, and photographed. To quantify neovascularization, the hemoglobin concentration in Matrigel homogenates was measured using the QuantiChrom Hemoglobin Assay Kit (DIHB‐250; BioAssay Systems). Formation of microvessels in the plugs was also assessed by CD31 immunofluorescence and hematoxylin and eosin (H&E) staining. Briefly, Matrigel plugs were embedded in OCT compound (Sakura Finetek, 4583) and cut into 5‐μm‐thick sections. After blocking, the sections were incubated with CD31 antibody (Abcam) followed by FITC‐labeled secondary antibodies (Abcam) for 30 min. Images of the sections were examined at a magnification of ×100 under an Invitrogen EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific). Sections with a thickness of 5 μm were also stained with hematoxylin and eosin (H&E). This work was approved by the Animal Care Committee of Guangdong Medical University and adhered to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication, 8th Edition, 2011).

BrdU (5‐bromo‐2‐deoxyuridine) incorporation assay was performed to evaluate the capability of cell proliferation. Briefly, cells were incubated for 1 h with 40 μM BrdU in normal medium at 37°C and then fixed with 4% paraformaldehyde. The fixed cells were treated with 0.05% trypsin to permeabilize them, followed by incubation with 3% BSA for 1 h at room temperature or overnight at 4°C. Cells were further incubated with a mouse anti‐BrdU monoclonal antibody (Cell Signaling Technology), followed by an Alexa Fluor 488‐conjugated secondary antibody (Cell Signaling Technology). Finally, cells were stained with DAPI (Sigma‐Aldrich) as a counterstain, and images were captured at 100× magnification under an Invitrogen EVOS FL Auto Cell Imaging System (Thermo Fisher Scientific).