Phospho smad3

Kidneys were fixed with phosphate buffered saline containing 4% paraformaldehyde for overnight and then embedded in paraffin. Sections (4 µm) were cut and deparaffinized in xylene, followed by rehydration in a graded series of ethanol. Staining was performed using hematoxylin and eosin, periodic acid Schiff (PAS), and Sirius red staining. Immunohistochemical staining was performed as described previously [24 (link)], using type I collagen (Abcam, Cambridge, UK), fibronectin (BD Biosciences, San Jose, CA, USA), β-tubulin (Abcam), phospho-Smad3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), TGF-β (Santa Cruz Biotechnology), and insulin (Santa Cruz Biotechnology). Thickness of the glomerular basement membrane (GBM) was measured by electron microscopy (H-7100; Hitachi, Tokyo, Japan). Renal fibrotic areas were quantified by morphometric analysis using a light microscope equipped with an imaging system containing aMRc5 Carl Zeiss microscope (Oberkochen, Germany) and iSolution DT version 7.7 software (IMT i-Solution, Coquitlam, BC, Canada). Areas of positive PAS matrix, Sirius red, immunostaining for type I collagen, and fibronectin in the renal fibrotic regions (brown color) were quantified by computer-based morphometric analysis. All data were normalized to the control and expressed as fold increase relative to the control.