Hematoxylin and eosin

Mouse lung tissues were fixed with 4% paraformaldehyde (Servicebio, Wuhan, China), embedded in paraffin, and sectioned. Sections were stained with hematoxylin and eosin (Servicebio) and Masson’s dye solution (Servicebio). The Ashcroft score refers to the methodology of Ashcroft et al., which is the average of the fibrosis scores of hematoxylin and eosin (HE)-stained sections assessed by two researchers for each animal^{35 (link)}.

Heart tissues were harvested after 4 weeks. Then, the heart tissues were fixed in 4% paraformaldehyde (pH 7.4). After fixation and paraffin embedding, the cardiac tissues were cut into 5-μm-thick sections. The sections were stained with hematoxylin and eosin (Servicebio; China) to analyze the global heart morphology and inflammatory cell infiltration. The sections were stained with Masson’s trichrome (Servicebio; China) to evaluate cardiac fibrosis. The sections were scanned at 20-fold magnification on a high-resolution microscope (Leica; Japan).

Heart samples were fixed in 10% buffered formalin solution and then embedded in paraffin. The samples were sectioned (5 μm thick) and then stained with hematoxylin and eosin (Servicebio, Wuhan,China) according to the manufacturing instructions, and the degree of inflammation was determined under 200 × magnification light microscopy. Myocardial histopathologic findings were quantified and scored for severity as follows: 0 = no inflammation, 1 = one to five distinct mononuclear inflammatory foci with 5% involvement or less of the cross-sectional area, 2 = more than five distinct mononuclear inflammatory foci or over 5% but not exceeding 20% involvement of the cross-sectional area, 3 = diffuse mononuclear inflammation involving over 20% of the area without necrosis, and 4 = diffuse inflammation with necrosis. The analysis was performed in a double-blinded manner by a trained pathologist. Besides, myocardial fibrosis was determined by Masson staining (Servicebio, Wuhan,China) according to the manufacturing instructions. Myocardial cells were stained red and collagenous fibers were stained blue. Collagen area fraction (collagen area/field area × 100%) was calculated by the Image-Pro Plus analysis system.