Mouse heart slicer matrix

Mice were fasted for 4 h and put in deep anaesthesia with a mixture of 4–5% isoflurane and O₂. Arterial blood was collected (by a small incision of the carotid artery) into tubes containing 50 μl of 0.5 M EDTA. Plasma was prepared by centrifugation at 500 × g for 20 min and 4 °C, snap-frozen in liquid N₂ and stored at − 80 °C. The heart was extirpated and separated into left ventricle (LV) and right ventricle, together with lungs and liver, rinsed in saline solution, blotted dry and weighed. A standardized 2 mm slice was taken from the LV using a mouse heart slicer matrix (Zivic Instruments, Pittsburgh, PA, USA). The heart slice was fixated in 4% formalin and embedded in paraffin. Remaining tissue was snap-frozen in liquid nitrogen and stored at − 80 °C^{22 (link)}.

Evaluation of infarct size was performed as previously described [17] (link). Briefly, after 24 hours of reperfusion, mice were anesthetized, the thoracic cavity opened and the slipknot re-ligated. The aorta ascendens was subsequently clamped and approximately 0.2 ml 2% Evans blue dye was injected into the proximal portion of the aorta, this allowing coronary perfusion and visualization of the area at risk (AaR). Hearts were subsequently excised, let to freeze on dry-ice before cross-sectioned into standardized 1 mm slices by the use of a mouse heart slicer matrix (Zivic Instruments, Pittsburgh, PA, USA). The slices were further incubated for 15 min in 1% 2,3,5-triphenyltetrazolium chloride (TTC, Sigma Aldrich, St. Louis, MO, USA) solution at 37 °C for visualization of viable myocardial tissue. Processed sections from CpG B injected animals (n = 25) and PBS injected animals (n = 35) were digitally photographed and analyzed for the extension of AaR (the non-Evans blue stained portion of the myocardium) and necrosis (the non-TTC stained portion of the AaR), using Adobe Photoshop (Adobe Systems Incorporated, San Jose, CA, USA). An investigator blinded for previous the intervention performed photograph analysis.

To evaluate the infarct size on day 1 post-MI, the hearts were weighed and frozen at −80 °C. The frozen hearts were cut transversely into 1-mm thick slices using a Mouse Heart Slicer Matrix (Zivic Instruments, Pittsburgh, PA, USA) and stained with 2% 2,3,5-triphenyltetrazolium chloride in PBS (pH 7.4) for 20 min in a 37 °C water bath. After fixation for 4 to 6 h in 10% neutral buffered formaldehyde, both sides of each slice were photographed. The viable myocardium was stained brick red, while the infarct tissues appeared pale white. Infarct and LV area were measured using automated planimetry with ImageJ software. The infarct size was then expressed as a percentage of the total LV area.