Accustain trichrome stains masson

Serial 10 μm cryosections were cut distally from the aortic root. Sections were stained (200, 400, and 600 µm after the appearance of the aortic cusps) with Oil Red O (Sigma-Aldrich, St. Louis, MO, USA) and Masson´s trichrome (Accustain Trichrome Stains–Masson; from Sigma-Aldrich). For immunohistochemical staining of macrophages, aortic root cryosections (160 μm from the aortic cusps) were incubated with rat anti-mouse Mac-2 antibody (1:1000; Cedarlane, Hornby, BC, Canada) or isotype control antibody (1:1000; Biolegend, San Diego, CA, USA), followed by horseradish peroxidase–conjugated goat anti-rat IgG secondary antibody (1:1000; GE Healthcare, London, United Kingdom) and visualized with the DAB substrate kit (Dako, Glostrup, Denmark). The areas of Oil red O staining, Mac-2 staining, and collagen staining (blue color in Masson’s trichrome) were determined using morphometric analysis (BioPix Software) and normalized to the size of the atherosclerotic lesion. Lesion complexity (presence/absence of necrotic core) was evaluated in sections stained with Masson´s trichrome (200 μm from the aortic cusps).

Accustain Trichrome Stains (Masson) (Sigma-Aldrich, USA) and Sirus Red/Fast Green Collagen (Chondrex, USA) staining kits were used to visualize fibrosis in rat LV. Paraffin sections with 6 μm thickness were dewaxed and stained as per supplier’s instruction [27 (link)]. To assess the severity of fibrosis in LV myocardium, pictures of Sirus Red/Fast Green stained paraffin sections were analyzed. Images of cross-sectional myocardium were processed by an in-house C++ program. Captured images were stored in Joint Photographic Expert Group (JPEG) format with 8-bit depth and the pixel value in any color channel varied from 0 to 255. To quantify fibrosis (red color) in the entire LV wall, all pixels from the image was read and categorized into three labels: collagen (red), non-collagenous proteins (green), or background after excluding staining artifacts, LV epicardium, and right ventricular wall. Pixels containing all red, green and blue values higher than 255 were considered as background and excluded from analysis. Since collagen is stained as red, a pixel was defined as red when its value was 30 times higher than the green pixel. Finally, the total number of fibrosis pixels were divided by the sum of red and green pixels to obtain the percentage of fibrosis in LV.