Metamorph imaging software version 6.1

For quantification of mesangial extracellular matrix, 3 μm sections from paraformaldehyde-fixed, paraffin-embedded kidney slices were stained using Periodic Acid-Schiff’s reagent (PAS) (n = 5/group). Mesangial area was expressed quantitatively by calculating the percentage of the total glomerular area that was PAS positive. Fifteen glomerular tufts per animal were chosen randomly for analysis. Quantification of glomerular and PAS positive areas was performed with MetaMorph Imaging Software (version 6.1) (Molecular Devices Corporation, Downingtown, PA), calibrated for the microscope and digital camera used to capture the images.

Under general anesthesia, both kidneys were flushed under constant 100 mm Hg pressure with phosphate buffered saline (PBS) containing sodium heparin (50 U/ml) through a cannula placed in the abdominal aorta. The left kidney was ligated while the right kidney was perfused with a ferric oxide slurry for later isolation of glomeruli. The left kidney was removed and weighed. Kidney cortical regions were dissected and snap frozen in liquid nitrogen, or fixed overnight in a solution of 2% paraformaldehyde in PBS. Paraffin-embedded, paraformaldehyde-fixed tissue sections were stained with PAS (periodic acid-Schiff, immunostained with WT-1 antibodies to identify podocytes, and counter-stained with picrosirius red. Fifteen glomeruli per mouse were chosen randomly for quantification of glomerulosclerosis by proportionally scoring areas positive for PAS staining (MetaMorph Imaging Software version 6.1; Molecular Devices Corporation, Downingtown, PA, www.moleculardevices.com) and mesangial index [6 ].