Hematoxylin eosin he stain

Anterior leaflets of mitral valvetissue samples, from subjects undergoing valve replacement and from post-mortem control subjects without any known history of cardiovascular disease were collected and fixed in 10% formalin. Samples were then embedded in paraffin and 5 µm thick sections were prepared according to standard procedure. Tissue sections were either stained with hematoxylin - eosin (HE stain, Sigma Chemical Co., St Louis, MO, USA) as described earlier [18] (link) or with Masson Trichrome to examine fibrosis. Sections were examined under an Olympus BX51 (Olympus Corporation, Tokyo, Japan) microscope and images were captured with a digital camera attached to it. Picrosirius red -0.1% Sirius red F3B solution in picric acid (Direct Red 80; Sigma Chemical Co., St. Louis, MO, USA) staining was also performed in valve leaflets to examine the collagen content and alignment and infer type of collagen. Stained sections therefore were observed using a binocular polarized light microscope (Nikon Eclipse binocular polarising microscope, LV 100 POL, Japan) with the same exposure time for each section. Under polarized light, birefringence is specific for collagen where red colour shows fibrillar type I collagen and yellow green colour indicates reticular type III collagen [19] .

nHAC/PLA was provided by Tsinghua University Materials Department, China. Electronic balance, microscope, slicer, LEICA2500E non-decalcification slicer, and tungsten steel slicer were provided by the People’s Liberation Army General Hospital, China. DIA-2000 pathological image analyzer was provided by Wuhan University, China. Hematoxylin-eosin (HE) stain and Goldner’s trichrome stains were purchased from Sigma-Aldrich.