Renal histology analysis by H&E staining and periodic acid-Schiff (PAS) staining

One side of the rat kidney was fixed with 10% neutral buffered formalin (at room temperature ≥24 h), dehydrated with gradient alcohol (at room temperature, 75% x2, 30 min; 95% x3, 30 min; and 100% x2, 30 min), cleared with xylene, waxed, embedded and sectioned (3 µm). Tissue sections were deparaffinized in xylene and stained with H&E for 20 min at room temperature. For PAS staining, histological sections were deparaffinized, oxidized in 1% aqueous periodate solution for 15 min and washed three times in distilled water. Sections were then soaked in Schiff solution for 15 min. Then, the sections were rinsed under running water for 15 min. Subsequently, the nuclei were stained with hematoxylin (room temperature for 10 min) followed by differentiation with ethanol-hydrochloric acid. The areas of nuclei appeared blue, while the basal membrane of the glomerulus and renal tubules, cytoplasm, mesangial matrix of the glomerulus and collagen fibers were red under light microscopy (magnification, x200 or x400)(BX51; Olympus Corporation). The relative mesangial matrix index and the relative glomerular volume were calculated (18,19).