Qwin standard version 2

Transforming growth factor 1 (TGF-β1) localization was assessed by immunohistochemistry in 3 µm sections. Samples were deparaffinized, rehydrated and incubated at 98 °C, pH 6 (Dako EnVision Flex Retrieval Solution Low pH, Dako, Glostrup, Denmark) for 30 min and blocked with 3% of bovine serum albumin (BSA) in PBS for 1 h. After overnight incubation at 4 °C with primary antibody of transforming growth factor beta 1 (TGF-β1) (1:100, AB92486, Abcam, Cambridge, UK) in 3% BSA (Sigma-Aldrich, San Luis, CA, USA) in PBS, slices were washed and incubated with a biotinylated secondary antibody following the manufacturer’s instructions (Dako REAL EnVision, Glostrup, Denmark).
A negative control without a primary antibody was used to set the level of the lowest detectable staining intensity. Along with Masson’s trichrome staining, semiautomatic image analysis software (Leica QWIN standard version 2.3, Leica Microsystems, Wetzlar, Germany) was used. Briefly, the image of each heart was converted to grayscale; then, using the optical density function of the software, pixels that fell within a designed threshold were counted, obtaining a mean value of grey color density. TGF-β1 staining was expressed as the average optical density.

Morphological and histological changes in the heart, left ventricular (LV) wall, septum thickness and cardiomyocyte diameter were measured in deparaffined 3 µm sections. They were stained with hematoxylin-eosin (Sigma-Aldrich, San Louis, CA, USA) and visualized using an optical microscope (model DMRXA2, Leica Microsystems, Wetzlar, Germany) coupled to a digital video camera (model Dc-100, Leica Microsystems, Wetzlar, Germany). Captured images were evaluated using an image analysis system (Image J). The mean cardiomyocyte diameter was determined by measurement of transnuclear widths of random, longitudinally oriented in 20 myocytes with magnification 40×. The LV wall and septum thickness were measured using pre-design software, which pooled and analyzed a set of at least 50 blinded radius measurements from the center of the LV to its outer edge.
The myocardial total collagen area was determined by using Masson’s trichrome, using a semiautomatic image analysis software (Leica QWIN standard version 2.3, Leica Microsystems, Wetzlar, Germany). The measurements were blinded, and the results were expressed as percentages of the total myocardial area. The collagen fiber/muscular tissue ratio was calculated.