Tissue sections were dewaxed with xylene and rehydrated with graded ethanol. After boiling in Tris-EDTA buffer (10 mM Tris Base, 1mM EDTA Solution, 0.05% Tween 20, pH 9.0) for 25 min, sections were blocked in 10% normal goat serum with 1% BSA in TBS for 2 hrs at room temperature. Sections were stained with primary WT-1 (Wilms Tumor protein) antibody (Ab89001, Abcam, Cambridge, MA, USA) using ABC Kit (PV-9001, Zhongshan Goldenbridge, Beijing, China) that functions as podocyte marker.20 (link)
Figure 1 shows representative WT-1 stained glomerula. Analysis software (Image-Pro Plus 6.0), Media Cybernetics, Rockville, MD) generated a binary image in which the stained number, density and area could be automatically calculated as percentage of every glomerular area. Fifty fields per specimen were randomly selected that covered nearly the whole piece of cortex. Staining and scoring was performed by a single observer who was masked to slide identity.![]()
WT-1 stained glomeruli. Sections from kidney tissue s were stained with primary WT-1 (Wilms Tumor protein) antibody (Ab89001, Abcam, Cambridge, MA, USA) using ABC Kit (PV-9001, Zhongshan Goldenbridge, Beijing, China) that functions as podocyte marker 400×.