Masson s trichrome staining kit

The sections were stained with Masson’s Trichrome staining by following the protocol of Masson’s Trichrome staining kit (Bio-Optica, Milano, Italy). The Zeiss Axiophot microscope (Carl Zeiss AG, Werk Göttingen, Germany) was used to observe illumination through a bright light, and photographs were acquired by a Discovery C30 camera (Tucsen Imaging Technology Co., Ltd. Fujian, China).

PBS‐rinsed hearts were fixed in freshly made formalin (Sigma‐Aldrich) overnight at 4°C and then processed with four cuts parallel and transversal to the tip, the midline of the ventricles, the base, and the atria. After processing, the tissues were embedded in paraffin (Leica Microsystems, Wetzlar, Germany); 5‐μm‐thick serial sections were deparaffinized, rehydrated, and stained with haematoxylin and eosin (Bio‐Optica, Milan, Italy). Heart horizontal sections at midlevel were also stained with Masson's trichrome staining kit (Bio‐Optica), following the manufacturer's protocol. Images were taken with Leica ICC50 microscope (Leica Microsystems) at 40× magnification. LAS AF Leica software (Leica Microsystems) was used for acquisition. Five images for sample (a total of 25 images for group of analysis) were used to quantify the Masson blue staining through ImageJ software.

WKY and SHR (n = 5/group) were anaesthetized and perfused with normal saline. Hearts were extracted, washed in PBS, fixed with 10% phosphate-buffered formalin and paraffin embedded, then 5 µm-thick sections were cut from each sample. Masson’s trichrome staining kit was used to perform the assay (Bio-Optica, #04-010802). For immunohistochemistry assays, formaldehyde-fixed paraffin sections were kept 35 min at 97.5 °C in 9 mM sodium citrate pH 6.0. Endogenous peroxidase activity was quenched with 3% H₂O₂ for 10 min; incubation of primary antibodies was performed overnight (O/N). Staining was performed with 3,3-diaminobenzidine (DAB) as a chromogen. Slides were immunostained in the same batch, including negative controls lacking the primary antibody. Antibodies raised against TGF-β1 (AbCam, ab64715, clone 9016) and ανβ5 (AbCam, ab179475, clone EPR16800) were used. As a negative control, species- and isotype-matched IgGs were incubated in place of the primary antibodies. Serial sections derive from comparable areas of the left rat heart ventricle, and were viewed with AxioSkop microscope equipped with AxioCam camera (Carl Zeiss) and analyzed with AxioVision 4.7 software (Carl Zeiss). The positive areas for TGF-β1 and ανβ5 on heart samples were normalized to the section area, calculated with AxioVision 4.7 software (Carl Zeiss).