Masson Trichrome Analysis of Cardiomyocyte Size

Four weeks after AntCaNtide (50 μg/kg), tat-CN17β (50 μg/kg) or NaCl 0.9%, intra-cardiac injection, the hearts were immersion fixed in 10% buffered paraformaldehyde. The tissues were embedded in paraffin, cut at 5 μm, and processed. For Masson trichrome staining of collagen fibers, slides were stained with Weigert Hematoxylin (Sigma-Aldrich, St. Louis, MO) for 10 min., rinsed in PBS (Invitrogen) and then stained with Biebrich scarlet-acid fuchsine (Sigma-Aldrich St. Louis, MO) for 5 min. Slides were rinsed in PBS and stained with phosphomolybdic/phosphotungstic acid solution (Sigma-Aldrich St. Louis, MO) for 5 min. then stained with light green (Sigma-Aldrich) for 5 min. Slides were rinsed in distilled water, dehydrated with 95% and absolute alcohol and a coverslip was placed. For the analysis of cardiomyocytes size, Masson trichrome staining sections were used [37 (link)]. The areas (μm²) of ~100 cardiac myocytes per heart were measured with the public domain Java image processing program Image by an independent operator blind to the study protocol (DS) [44 (link)].