Histological Analysis of Cardiac and Renal Fibrosis

Tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned to 5 µm thickness. The heart and kidney were stained with a Trichrome Stain (Masson) Kit (Sigma, St. Louis, MO, USA) to determine collagen fibrosis. The aorta was stained with an Elastic Stain Kit, a modified Verhoeff Van Gieson Elastic Stain Kit (Sigma, St. Louis, MO, USA). Quantification was performed in 3 random fields of view per section with an optical microscope (×400 magnification). Renal and cardiac perivascular fibrosis was analyzed/graded from 0 to 4 by using a histology damage score (0, no lesion; 1, focal lesion; 2, multifocal mild lesion; 3, multifocal moderate lesion; and 4, diffuse, moderate, or severe damage) [35 (link)]. The percentage of tubulointerstitial damage and vascular elastin distribution in the aorta were quantified by ImageJ software [36 (link), 37 ].