Ab2350

Mouse skin tissues were fixed with 4% paraformaldehyde for overnight. Then, the tissues were washed, sealed with normal saline of 0.01 M phosphate buffer for three times, and then sealed with 10% goat serum (C0265, Beyotime, Shanghai, China) at room temperature for 30 min. After that, transforming growth factor-β1 (TGF-β1) (1:200, ab92486, Abcam), vascular endothelial growth factor (VEGF) (1:200, ab2350, Abcam), and platelet-derived growth factor (PDGF-BB) (1:200, ab9704, Abcam) were incubated with tissues at 4 °C for overnight. The secondary antibody and 4′,6-diamidino-2-phenylindole were incubated at room temperature in the dark for 1 h, and glycerol was fixed. The confocal laser scanning microscope was used for analysis (LSM, FV1000; Olympus Corp., Tokyo, Japan).

The selected sections were deparaffinized, blocked and incubated with primary antibodies against BMP2 (Abcam, Cat#AB214821), OCN (Abcam, Cat#AB93876), VEGFR-1 (Abcam, Cat#AB2350), and CD31 (Abcam, Cat#AB32457) at a 1:200 dilution by 3% w/v BSA overnight at 4 °C. After washing, all samples were incubated with horseradish peroxidase-conjugated secondary antibodies (ZSGB-BIO, diluted with PBS, Cat#PV-9001) and observed under a Zeiss light microscope. Each group comprised at least 3 slides, and each slide was observed at the defect margin and the defect center including the scaffolds.

As previously described, immunofluorescence analyses were performed ( 19 (link) ). Frozen ovarian tissue sections were air-dried for 30 min at room temperature. These sections were washed with PBS twice for 5 min each and incubated with 2.5% normal goat serum (Vector, S-1012) for 1h at room temperature in a humidified chamber. Subsequently, these sections were incubated overnight at +4°C with VEGF (Abcam, ab46154, dilution; 1/100), VEGFR1 (Abcam, ab2350, dilution; 1/100) and VEGFR2 (Abcam, ab39256, dilution; 1/500) antibodies. The next day, the sections were incubated with secondary antibodies for 45 min in darkness.

Tissues underwent antigen retrieval by boiling in an electric pan with 10 mM citrate buffer, pH 6.0, for 40 min. Endogenous peroxidase blockade was done with 3% hydrogen peroxide in phosphate-buffered saline (PBS). Nonspecific binding sites were blocked with 10% goat serum diluted in PBS for 1 h in a humid dark room. The samples were then incubated overnight at 4°C for 12 h with the primary antibodies diluted in 1.8% bovine serum albumin (BSA) (VEGF: mouse, 1:150, ab1316, Abcam, USA; VEGFR-1: rabbit, 1:50, ab2350, Abcam, USA; VEGFR-2: mouse, 1:25, sc6251, Santa Cruz Biotechnology, USA). After removal of the primary antibody, the appropriate secondary antibody (VEGFR-1: biotinylated goat anti-rabbit-sc-2040 antibody, Santa Cruz Biotechnology; VEGF and VEGFR-2: biotinylated goat anti-mouse antibody-sc-2005, Santa Cruz Biotechnology) diluted 1:200 in BSA was added. For the negative control, the primary antibody was omitted. The samples were then incubated for 30 min in streptavidin-HRP (Biolegend #405210, USA) diluted 1:200 in PBS. Next, they were developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB, Sigma, USA) diluted 1:100 in hydrogen peroxide for 20 min. Finally, the samples were counterstained with Harris hematoxylin, washed in running water, dehydrated in an alcohol series and xylene, and covered with coverslips mounted with Permount^® (Fisher Scientific).