488 or 594 conjugated species specific secondary antibody

For histological evaluation, samples were fixed in 10% paraformaldehyde at 4°C overnight, then dehydrated in a graded series of ethanol and permeabilized in dimethyl benzene and embedded in paraffin wax. After the paraffin embedding, 4-μm thick sections were consecutively cut, and stained with H&E to analyze the morphology and microstructure. For DNA content evaluation, the sections were then counterstained with DAPI. Then, the sections were observed with immunofluorescence to detect the basement membrane proteins, which are composed of ECM components including laminin, collagen IV, and fibronectin.
For immunofluorescence, tissue sections were deparaffinized, and rehydrated, and antigen retrieval was performed. Sections were then blocked with 10% fetal bovine serum (FBS) for 30 minutes at room temperature. Primary antibodies were applied and left to incubate overnight at 4°C, followed by 488- or 594-conjugated species-specific secondary antibody (Chemicon, USA) at 1:400 dilutions for 2 hour at 37°C. Lastly the sections were rinsed in PBS for 45 minutes, and stained with DAPI. Slides were observed using an Olympus fluorescent microscope and images were taken using Olympus soft image viewer.

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).