Visualizing Capillaries and Fibrosis in Tissue

Tissue was prepared for histology, as described in [37 (link)]. Tissue sections 10 μm thick were plated on microscope slides and fixed in acetone for 5 min on ice. Slides were incubated for 1 h with fluorescein-conjugated lectin from Griffonia simplicifolia (Vector Laboratories, Burlingame, CA, USA), at a final concentration of 10 µg/mL in phosphate-buffered saline. Slides were imaged on a Nikon Eclipse E600 microscope with a Nikon Plan Fluor 20X objective, using 487 ± 10 nm excitation light and fluorescence collected at 533 ± 20 nm. Capillaries were identified by the bright focal staining pattern and their location digitized. Muscle fiber boundaries were identified by the less intense staining of the glycocalyx surrounding the cell membrane. Fibrosis was assessed using picrosirius red staining, as described previously [29 (link)]. Results from two to five sections per heart were averaged to calculate the representative value for each animal, which was used for subsequent statistical analysis.