Histopathology, Immunohistochemistry, and Immunofluorescence

Paraformaldehyde-fixed myocardial tissues were treated with gradient alcohol dehydration and then embedded in paraffin. Hematoxylin and eosin (HE) and immunohistochemistry staining was implemented in 4 µm thick sections of heart tissues. Immunofluorescence staining, fixation and permeabilization were performed according to routine procedures. Tissue sections were blocked in 5% normal goat serum for 1 h at room temperature and then incubated with primary antibodies of interest at 4°C overnight. The primary antibodies used for immunohistochemistry and immunofluorescence staining are as follows: VE-cadherin (1:100, Bioss, bs-0878R), F4/80 (1:100, Bioss, bs-11182R), CD31 (1:100, Abcam, ab24590), VCAM-1 (1:500, Abcam, ab134047), ICAM-1 (1:500, Abcam, ab171123), cTnT (1:400, Abcam, ab45932), and plasma Albumin (1:1,000, Abcam, ab8940). Nuclei were counter-stained with DAPI. At least five fields per rat with a high-density area in each group were randomly scored with the case Viewer Software (3D HISTECH) using a ×40 objective.