Mouse anti vegfr2

Samples were prepared for histological and immunofluorescence analyses by following standard protocols for paraffin embedding. For histological analysis, mounted kidney sections were stained by hematoxylin and eosin.
For immunofluorescence staining, the sections were blocked with 5% normal bovine serum in PBS for 30 min. Sections were then incubated with primary antibodies overnight at 4°C. The primary antibodies used were as follows: mouse anti-VEGFR2 (1:200; Abcam, UK), rabbitanti-CD133 (1:200; Abcam), goat anti-fibronectin (1:200; Abcam) and rabbit anti-CD31 (1:200; Abcam). After being washed in PBS, sections were then incubated in 488- or 594-conjugated species-specific secondary antibody (1:100; Chemicon, USA) for 2 hours. Slides were observed with an Olympus fluorescent microscope and images were captured using Olympus soft image viewer. The intensity of immunoreactivity was quantified using the Image-Pro Plus 6.0 software (Media Cybernetics, USA).

The cold RIPA buffer (Thermo Scientific, Rockford, IL, USA) was utilized for cell lysis. Minute (TM) Cytoplasmic and Nuclear Fractionation kit (SC003) was used to extract nucleoprotein. Thereafter, we used the BCA Protein Assay kit (Beijing Solarbio Science & Technology Co., Ltd., china) for quantifying protein content. After separating proteins through SDS-PAGE, they were transferred on the PVDF membranes. Then, each membrane was subjected to overnight incubation using primary monoclonal antibodies, including mouse anti-VEGFR2, rabbit anti-plexinA1 and mouse anti-gapdh (all Abcam, Cambridge, MA, UK), rabbit anti-Histon H3, rabbit anti-JAK, rabbit anti-STAT3, rabbit anti-p-JAK and rabbit anti-p-STAT3 (all ABclonal Co., Ltd,Wuhan, China) as well as mouse anti-β-actin (Cell Signaling Technology, Danvers, MA, USA) antibodies under 4°C. Afterwards, HRP-labeled secondary antibodies (Santa Cruz, CA, USA) were further used to incubate the membranes for 2 h. In addition, the enhanced chemiluminescence (ECL) substrate (Pierce, Rockford, IL, USA) was used for detecting protein blots, whereas the Image analysis software was employed for visualization.