Ab2349

Tissues were sectioned to a thickness of 5 μm for immunostaining. Each slide was deparaffinized in xylene and rehydrated through a graded alcohol series. For antigen retrieval, sections were heated in 0.01 M sodium citrate buffer (pH 6) using a pressure cooker for 20 min, then cooled to room temperature for 10 min and washed with TBST for 3 min. Subsequently, to block nonspecific antibody binding, the sections were incubated with 5% normal goat serum for 1 h at room temperature (approximately 23 °C). The tissue sections were then incubated with primary antibodies, including anti-cluster of differentiation (CD31) (1:50 ab28364, Abcam, Boston, MA, USA ) and anti-CD34 (1:500, ab81829, Abcam) to identify vascular endothelial cells, anti-Ki-67 (1:500, ab15580, Abcam) to identify proliferating cells, and vascular endothelial growth factor receptor 2 (VEGFR2) (1:250, ab2349, Abcam) to identify VEGF-responsive cells for 1 h at room temperature in a humidified chamber, except for VEGFR2, which was incubated overnight at 4 °C. Following primary antibody incubation, sections were washed with TBST for 3 min. The sections were then incubated with an Alexa Fluor™ 633 goat anti-rabbit secondary antibody for 1 h at room temperature. Finally, nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI 1%, BIOTIUM, Fremont, CA, USA) for 5 min.

EPCs were fixed with 40 g/L polyformaldehyde for 15 min, rinsed with PBS three times, blocked with 10% goat serum, and incubated with the primary antibodies CD34 (ab110643, Abcam Inc., Cambridge, MA, USA), CD133 (ab222782, Abcam), vWF (ab6994, Abcam), and VEGF-2 (ab2349, Abcam) at 4 °C overnight. Then, the cells were added with TRITC-labeled fluorescent secondary antibody goat anti-rabbit IgG H&L (ab6718, 1:1000, Abcam) and FITC-labeled fluorescent secondary antibody goat anti-mouse IgG H&L (ab6785, 1:1000, Abcam) for 45 min. Following two PBS washings, the cells were cultured with Hoechst3342 for 15 min, washed, and finally photographed using a fluorescent microscope.

Antibodies against CD31 and VEGFR2 were purchased from Abcam (Cambridge, UK). Immunohistochemical staining procedures were performed by Pfizer (Groton, CT) and at the Brigham & Women’s Hospital (Boston, MA). E14.5 has been established as the optimal time point for mouse developmental disorder phenotype analyses^{18 (link)}. In short, tissues were collected immediately after E14.5 embryo collection, fixed in 10% buffered formalin and embedded in paraffin. Four micrometer serial sections were cut and mounted on glass slides. One section from each lesion was stained with hematoxylin and eosin (H&E). Other slices were stained for CD31 (ab28364) or VEGFR2 (ab2349) with hematoxylin counterstaining following Abcam’s protocols.

Dewaxing to water, the slice soaked in 3% hydrogen peroxide solution after 15 min, PBS cleaning section, 0.1 M sodium citrate solution for antigen after repair, add VEGF (dilution as 1:100, Abcam, ab53465), VEGFR1 (dilution as 1:250, Abcam, ab32152), VEGFR2 (dilution as 1:100, Abcam, ab2349) antibody, 4°C incubation overnight, PBS cleaning slice, add 2 resistance of biotin labeling, 37°C after incubation for 30 min, PBS cleaning slice, join the DAB chromogenic liquid color, hematoxylin dyeing redyeing nuclei, neutral resin sealing film, were observed under microscope.