Rabbit anti rat flk 1

The HA/(COL/HEP)₅/VEGF/MSCs and HA/VEGF/MSCs constructs were incubated in α-MEM culture medium containing 10% FBS and were cultured at 37 °C with 5% CO₂. The constructs were collected after 7 d and evaluated to determine the expression of the rat CD31, Flk-1, and von Willebrand factor (vWF) mRNAs and proteins using real-time quantitative polymerase chain reaction (qRT-PCR), immunofluorescence, and western blotting (see below), respectively.
For the immunofluorescence assay, the cells were fixed in 4% paraformaldehyde for 15 min and then blocked with 1% BSA for 20 min at room temperature. After an overnight incubation with the mouse anti-rat CD31 antibodies (Abcam, UK) and rabbit anti-rat Flk-1 (Abcam) antibodies at 4 °C, the cells were incubated with the appropriate secondary antibodies (AlexaFluor 594-conjugated goat anti-mouse or AlexaFluor 488-conjugated goat anti-rabbit; Invitrogen, USA) for 1 h at 37 °C. After washing with PBS, the samples were observed under CLSM. DAPI staining (blue) was applied to highlight the total nuclei. Six locations per scaffold were randomly photographed, and the relative fluorescence intensity per cell was determined using the ImageJ software.

The in vitro and in vivo expression of the rat CD31, Flk-1, and vWF proteins was assessed by western blotting. Seven days after implantation, the samples were harvested and stored in liquid nitrogen until evaluation. The frozen samples were completely homogenized in RIPA lysis buffer (50 mM Tris-HCl, 0.1% Triton X-100, 2 mM ethylenediaminetetraacetic acid (EDTA), 100 mM NaCl, and 1 mM protease inhibitors) for 30 min at 4 °C and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
After being transferred to a poly(vinylidene fluoride) membrane (Millipore, MA, USA), the membranes were incubated with a solution of 5% milk powder in Tris-buffered saline (TBS) to prevent nonspecific interactions. Then, the membranes were incubated overnight with the antibodies and detected using an ECL (ECL western blotting substrate, Pierce, USA) system. The specific primary antibodies used for this experiment were mouse anti-rat CD31 (Abcam, UK), rabbit anti-rat Flk-1 (Abcam, UK), and mouse anti-rat vWF (Santa Cruz, USA) antibodies.