Masson ht15 1kt

Isolated hearts were fixed in 10% formalin, dehydrated and paraffin embedded. The 5 μm sections were cut and stained with hematoxylin and eosin (H&E) and wheat germ agglutinin (WGA, Alexa Fluor 488 conjugate; Thermo Fisher, Rockford, IL, USA). Masson (HT15‐1KT; Sigma‐Aldrich, St. Louis, MO, USA) staining was performed to determine collagen deposition following the manufacturer's instruction. The slides were examined microscopically and the fibrotic area was determined by the area of myocardial collagen/the area of the field using Image Pro‐Plus version 6.0 (Media Cybernetics, Rockville, MD, USA) image analysis software.

The isolated hearts from mice were fixed with 10% formalin, dehydrated, and embedded in paraffin. Five histopathology sections were acquired and stained with hematoxylin-eosin (H&E) and wheat germ agglutinin (WGA, Alexa Fluor 488 conjugated). The cross-sectional area of cardiomyocytes was determined via Thermo Fisher staining. Collagen deposition was evaluated by Masson (HT15-1KT, Sigma Aldrich) staining kit following the manufacturer's protocol. Section microscopy was performed. The fibrosis area based on myocardial collagen area/visual field area was determined using the Image Pro-Plus 6.0 Image analysis software.

For histological analyses, mouse hearts were fixed in situ by retrograde perfusion with PBS (pH 7.4) containing 50 mM KCl and 200 U/mL heparin for 2 minutes at 80 mm Hg, followed by in situ paraformaldehyde fixation. Sections were embedded in paraffin and stained with H&E and wheat germ agglutinin (WGA, Alexa Fluor 488 conjugate; Thermo Fisher). Masson (HT15‐1KT; Sigma‐Aldrich) staining was performed to determine collagen deposition following the manufacturer's instruction. Tissue morphometry was performed in a blinded fashion using the Quantimet 500MC digital image analyzer.