Quantitative Cardiac Tissue Analysis

Paraffin-embedded LV sections were treated as described in the previous section for deparaffinization, antigen retrieval, and nonspecific binding blockade. Subsequently, samples were incubated overnight at 4 °C with a vimentin primary antibody (Ab92547 Abcam, Cambridge, UK; 1/150 dilution) and the next morning with the secondary antibody goat antirabbit IgG conjugated to Alexa Fluor 405 (A31556 Thermo Fisher, Waltham, MA, USA; 1/200 dilution) at room temperature for 120 min. Additionally, samples were labeled with a wheat germ agglutinin (WGA) lectin conjugated to Alexa Fluor 594 (W11262 Thermo Fisher; 1/200 dilution) and with isolectin GS-IB4 conjugated to Alexa Fluor 488 (I21411 Thermo Fisher; 1/200 dilution) for 120 min. Sections were mounted with Fluoromount (Sigma-Aldrich, St. Louis, MO, USA). Microphotographs were obtained in three different regions at the three different bandpass filters (345–445, 470–525, and 550–605) for both LV and IVS at a magnification of 63×. Myocyte count, cross-sectional area, extracellular matrix content (ECM), capillary, and fibroblast count were assessed with morphological measurements with ImageJ [13 (link)].