Microfire digital camera

Hearts were fixed with 10% zinc-formalin (Thermo Fisher Scientific, Waltham, MA) as previously described [27 (link)]. Sections were stained with hematoxylin-eosin or Masson’s trichrome stains. Imaging was performed using an Olympus LX81 inverted light microscope (New York, NY). Cardiomyocyte cross-sectional area and left ventricular thickness were quantified from images taken by an Olympus Microfire Digital Camera as described [28 (link)].

Myocardial fragments were stained by Masson's trichrome for visualization and quantification of fibrotic tissue. Light microscopy was performed using an Olympus TH4-100 inverted microscope that was connected to an Olympus Microfire Digital Camera (New York, NY). For quantification of fibrotic tissue, pictures from each heart section (thickness of 3 μm) were taken systematically to ensure that the entire extent of the tissue section had been covered. Five different hearts were used per group, and a total of 20 sections were analyzed using the Image Pro-plus software (Media Cybernetics, Silver Springs MD). The percentage of fibrotic tissue in each micrograph was measured and averaged per heart. The average value for each heart was then utilized for statistical analysis. Vascularization Index (number of vessels/mm²) was determined in endothelin-1 immunostained sections of the left ventricle of mice from all groups. The number of capillaries per area was determined in 50 random fields per group, following the “Forbidden Line Principle” [13 (link)].

RNA in situ hybridization was performed as described (Feng et al, 2010 (link); Prince et al, 1998 (link)). The probes used in this study: bozozok, chordin, eve1, gsc, egr2b (krox20), myoD, pax2a and squint (ndr1) were obtained from Dr. Solnica-Krezel (Washington University, Saint Louis, USA). Unless otherwise stated, images of whole mount embryos were acquired using an Olympus SZ61 dissecting microscope equipped with a high-resolution digital camera (model S97809, Olympus America) and Picture Frame 2.0 or 3.0 software (Optronics). Two-color in situ hybridization embryos were flat mounted as described and images were acquired using an AxioPlan2 microscope equipped with an AxioCam CCD camera (Zeiss). Images of fluorescent in situ hybridization embryos were acquired using Leica SP5 confocal. GFP reporter expression of the Tp1bglob:egfp line was acquired using an Olympus SZ16 fluorescent dissecting microscope and Microfire digital camera (Olympus). Images were processed in ImageJ, Adobe Photoshop, and Adobe Illustrator.