Cardiomyocytes hypertrophy, interstitial fibrosis and immune cells infiltration
Whole hearts and ventricles were harvested, rinsed of blood with pre-chilled PBS and were fixed in 3% formalin. The heart specimens were then embedded in paraffin and sliced into 4μm thick sections. Haematoxylin and eosin (H&E), Masson’s trichrome, and CD68 immunohistochemical (IHC) stainings were performed on tissue sections. The diameter of cardiomyocytes from the H&E stained sections was measured to determine the increase in myocyte size. The trichrome stained sections were used to evaluate the percentage of collagen deposit by dividing the collagen area with the total myocardial area and multiple by 100, as described earlier (Jia D et al., 2019).
CD68 IHC staining was done following the general protocol with a few optimizations. In brief, after deparaffination, heat-induced antigen retrieval was done using citrate butter. Non-specific antibody binds were prevented by flooding the slides with H2O2 for 10 min and followed by 3% BSA for 30 min. The tissue sections were then incubated overtime at 4°C with CD68 primary antibody (ab955, Abcam) diluted in 1% BSA. The tissues were incubated with biotinylated goat anti-rabbit IgG and Streptavidin peroxidase for 25 min each, followed by DAB staining and hematoxylin counterstaining. Imaging of tissue slides was done at x400 magnification and analyzed with ImageJ (1.52a version; National Institute of Health, USA) was utilized in these analyses.