Vegfr2

Rats’ right middle lung lobes were isolated and fixed in 4% paraformaldehyde for 24 h. Five μm thick paraffin sections where processed and stained with H&E and evaluated under an optical microscope (DMI3000B; Leica, Wetzlar Germany). Antigen retrieval for immunohistochemistry was carried out after dewaxing and hydration of the sections by heating in 500 ml of 10% citric acid buffer (pH 6.0) for 10 min. An endogenous peroxidase blocker (ZSGB-BIO, Beijing, China) was next added for 10 min at room temperature, following by incubation with normal goat serum working solution (ZSGB-BIO, Beijing, China) for 10-15 min at room temperature. Then, antibodies against occludin(1:200, rabbit lgG; Abcam, USA), notch1(1:400, rabbit lgG; Proteintech Group, Inc., Chicago, IL), CD31(1:2000, rabbit lgG; Abcam, USA), Hes-1(1:200, rabbit lgG; Abcam, USA), VEGF-A (1:200, mouse lgG; Abcam, USA) and VEGF-R2 (1:250, rabbit lgG; Abcam, USA) were applied and incubated at 4°C overnight. Biotinylated goat anti-rabbit/mouse IgG (ZSGB-BIO, Beijing, China) and HRP-conjugated streptavidin/avidin antibody working solutions (ZSGB-BIO, Beijing, China) were added with 10-15 min incubations at room temperature. Freshly prepared DAB solution (Solarbio, Beijing, China) was used for signal detection, followed by counterstaining with hematoxylin.

The grafted fat paraffin-embedded sections were stained with hematoxylin, eosin, and immunohistochemistry. The sections were primarily incubated with rabbit anti-human and mouse CD34 (Abcam, USA), VEGFR2 (Abcam, USA), and Ki-67 (Abcam, USA) separately, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Finally, the staining color was developed using the DAB Detection Kit (Maixin, Fuzhou, China). Histologic parameters were examined under a light microscope. HE staining was observed in 5 low magnification field analysis methods for the assessment of fat graft integrity, as evidenced by the presence of intact and nucleated adipocytes and the presence of cysts and vacuoles. Each parameter was graded by two observers independently on a semiquantitative scale ranging from 0 to 5 (0 = absence; 1 = minimal presence; 2 = minimal to moderate presence; 3 = moderate presence; 4 = moderate to extensive presence; and 5 = extensive presence).

Total proteins extracted from cells or tumor tissues were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the proteins in the gel were electrotransferred onto polyvinylidene fluoride membranes. The proteins were then probed with antibodies against DR6 (BioVision, Milpitas, CA, USA), IL-6R (Proteintech, Rosemont, IL, USA), VEGF-R-2 (Abcam, Cambridge, MA, USA), VEGF-D and PDGFR-α (Biogot, Nanjing, China), caspase 3, caspase 6, p-IκBα, p-P38, P38, p-STAT3 or STAT3 (Cell Signaling Technology, Danvers, MA, USA) and then incubated with an appropriate horse radish peroxidase (HRP)-conjugated secondary antibody (Cell Signaling Technology). The target proteins were visualized with a chemiluminescence system (Millipore, MA, USA) and exposed to X-ray film. The blot images were scanned and processed using Microsoft Office Picture Manager. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (KangChen Biotech, Beijing, China) was used as an internal control.

An equal amount of protein from each sample (35 μg) was loaded. Protocols previously published were used 31. Membranes were incubated overnight at 4°C with antibodies against VEGFR‐1, VEGFR‐2 (1:500; Abcam, Cambridge, MA, USA), VEGF (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and β‐actin (1:500; Santa Cruz Biotechnology) or α‐tubulin (1:500; Santa Cruz Biotechnology) as loading controls for cells. Finally, membranes were incubated for 2 hrs at RT in antimouse and anti‐rabbit IgG‐HRP (1:10,000; Santa Cruz Biotechnology). Bands were visualized with ECL (Pierce, Thermo Scientific, Rockford, IL, USA) and detected with Image Quant LAS‐4000 mini (GE Healthcare, Uppsala, Sweden). Protein levels were quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA). Protein expression intensity was normalized to the amount of loaded protein and plotted as a percentage of the control group.

MSCs cultured as above described were seeded on cover slips in 6-well plates. Briefly, After fixing and blocking, cells were labeled at 4°C overnight in the presence of an antibody directed against vWF (Dako Cytomation, Denmark),VEGFR1 (SigmaAldrich),VEGFR2 (abcam), and CD31 (abcam), respectively. Nuclei were labeled by adding 40-6-diamidino-2-phenylindole(Invitrogen,USA). Cells were photographed under a fluorescent microscope (Nikon, Japan).