Aperio scanscope at slide scanner

After echocardiography, whole hearts were harvested from the sacrificed rats and fixed with 10% formalin. The hearts were sliced into 3 sections along the transverse axis below the ligature to the apex at 2-mm intervals. The samples were dehydrated in a series of ethanol, embedded in paraffin, and cut into 4-μm-thick sections. The sections were deparaffinized in xylene, ethanol, and distilled water and stained with H&E and Masson’s trichrome to visualize cardiac fibrosis. Stained slides were scanned using the Aperio ScanScope AT Slide Scanner (Leica Biosystems Inc., Buffalo Grove, IL, USA) at ×200 magnification, and all images were captured using ImageScope software (Leica Biosystems Inc.). The infarction size and fibrosis area of the LV were determined using the following formula [13 (link)]: $$\begin{array}{c}\text{Infarction size}=\hfill \\ \left[\left(\text{epicardial circumference of the total LV area}\right.\right.\hfill \\ +\left.\text{endocardial circumference of the total LV area}\right)/\hfill \\ \left(\text{epicardial circumference of the total LV area}\right.\hfill \\ \left.\left.+\text{endocardial circumference of the total LV area}\right)\times 100\%\right]\hfill \end{array}$$ $$\begin{array}{c}\text{Fibrosis area}=\left[\left(\text{fibrosis area of LV}/\text{total area of LV}\right)\times 100\%\right]\hfill \end{array}$$
The LV wall thickness was measured in the infarcted and border zones. Each zone was divided into 6 equal segments, and LV wall thickness was calculated by averaging the thicknesses of the 6 segments [6 (link)]. All histological analyses were quantified using ImageJ software.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect apoptosis in formalin-fixed paraffin-embedded (FFPE) tissues. Irradiated tumor tissues were fixed with 10% neutral buffered formalin (NBF) for 4 h and embedded in paraffin. After deparaffinization, TUNEL staining was performed using In Situ Cell Death Detection Kit (Roche Diagnostics, Mannheim, Germany). Images were captured using an Aperio ScanScope AT slide scanner (Leica Biosystems Inc. Buffalo Grove, Illinois, USA) and analysed using ImageScope software (Leica Biosystems).

Deparaffinized and dehydrated tumor tissue sections were stained by the TUNEL In Situ Cell Death Detection Kit (Roche Applied Science, Mannheim, Germany) according to the manufacturer’s protocol. Briefly, tumor tissue sections were placed in a 3% hydrogen peroxide solution with methanol to block endogenous peroxidase activity and were incubated in 0.1% sodium citrate containing 0.1% Triton X-100 to increase tissue permeability. After rinsing in PBS, 50 µL of the TUNEL reaction mixture (calf thymus TdT and nucleotides) was added to each sample. After incubation at 37 °C in the dark for 60 min, these sections were rinsed with PBS and the apoptotic cells were marked by 3,3′-diaminobenzidine (DAB) through a horseradish peroxidase (HRP) catalysis of biotinylated dUTP-streptavidin-HRP. Images were captured using an Aperio ScanScope AT slide scanner (Leica Biosystems, Inc., Buffalo Grove, IL, USA). Numbers of TUNEL-positive cells were determined with ImageScope software (Version 12.4.6, Leica Biosystems, Inc.).