W11261 alexa fluor 488 conjugate

was performed using Thermo-Fisher #W11261 Alexa Fluor 488 conjugate. Sections on microscope slides were stained with 50 μg/ml WGA in PBS for 10 minutes at room temperature, followed by washing. In each heart, six images of CMs in transverse orientation were photographed at 400x magnification and processed using ImageJ software to calculate average pixel numbers/CM, as indicative of CM size. Briefly, the green (488) channel displaying CMs was isolated, followed by thresholding to fill-in spaces occupied by CM cytoplasm, then adjusting settings to acquire particle sizes in the 600−∞ range having a circularity of 0.25–1.0. After results (which were set to “include holes”) were obtained, particles representing incorrectly oriented CMs, and blood vessels, were removed. To assess CM density, the total number of CMs in each 400x microphotograph was manually counted by blinded observers and presented as the average number per 400x field.

WGA staining was performed using Thermo-Fisher #W11261 Alexa Fluor 488 conjugate and Thermo-Fisher #W11263 Alexa Fluor 350 conjugate. Sections mounted on microscope slides were stained with 50 μg/ml WGA in PBS for 10 minutes at room temperature, followed by thorough washing. Images of CMs in transverse orientation were photographed at 400x magnification and processed using ImageJ software to determine numbers of CMs and average numbers of pixels per CM as indicative of CM density and size. Briefly, the FITC (488) or DAPI (350) channel displaying CMs outlined in cross-section was isolated, followed by thresholding to fill-in spaces occupied by CM cytoplasm, then adjusting settings to acquire particle sizes in the 600-infinity range having a circularity of 0.25–1. After results (which were set to “include holes”) were obtained, particles representing CMs that were non-transversely sectioned, or blood vessels, were removed.