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.