P6744 1ga

Mid-papillary level transverse myocardial sections were deparaffinized and rehydrated as above. Sirius red/fast green stain was prepared using 0.1% (w/v) Direct Red 80 (Cat. 365,548–5G, Sigma-Aldrich) and 0.1% Fast Green FCF (w/v) (Cat. F7252, Sigma-Aldrich) in picric acid (P6744-1GA, Sigma-Aldrich). Myocardial sections were incubated in sirius red/fast green solution for 30 min at room temperature, rinsed in deionized water, dehydrated with 100% ethanol, rinsed in xylene, and mounted under glass coverslips using Permount Mounting Media (Cat.17986-05, Electron Microscopy Sciences). Brightfield images were digitally acquired using a Keyence BZ-X810 microscope using both 20 × and 4 × objectives.

Tissues were deparaffinized and rehydrated through graded ethanol series. Slides were incubated for 1 h at RT in Picrosirius red staining solution (1% (w/v) Direct Red 80 (2610‐10‐8; Sigma‐Aldrich) in saturated picric acid (P6744‐1GA; Sigma‐Aldrich)). The slides were washed twice with 0.5% acetic acid, dehydrated in graded ethanol series, cleared in Tissue Clear, and mounted with Pertex (Histolab, Cat # 00811). The slides were imaged with the Zeiss LSM800‐Airy confocal microscope using the 561 nm laser. Image quantification was performed with the macro TWOMBLI of Fiji‐ImageJ^[46] and the ridge detection plugin.

Paraffin-embedded lung sections from LFD- and HFD-fed mice were deparaffinized and rehydrated through alcohols. Slides were incubated for 1 hr in picrosirius red solution (0.5 g of Direct Red 80 (Sigma-Aldrich; 2610-10-8, St. Louis, MO, USA) in 500 mL of saturated picric acid (Sigma-Aldrich; P6744-1GA, St. Louis, MO, USA)). Slides were washed twice with acidified water (0.5% acetic acid) for 10 min, dehydrated in graded ethanol and xylenes, and mounted using Richard-Allan mounting medium (ThermoFisher Scientific, 4112APG, Waltham, MA, USA). Imaging of picrosirius red was performed using a Nikon Eclipse E600 Microscope and QICAM Fast 1394 camera. Collagen fluorescence was detected using a TRITC filter cube and images were taken at 200× magnification. Slides were blinded prior to imaging, and lung tissue areas of similar densities without large calibur vessels or bronchi were selected for quantification. To remove the autofluorescent background, images were captured using DAPI and FITC filter cubes at 200× magnification. ImageJ Image Calculator was utilized to subtract background autofluorescence from collagen fibers. After the background was removed, images were converted to 8-bit. Collagen fiber length, width, and number were measured using CT-FIRE detection software (LOCI; Madison, WI, USA) [35 (link)].