Masson trichrome staining

Mice were anesthetized with 1.0% inhaled isoflurane, and echocardiography was performed with a 12-MHz transducer (SONOS 7500; Philips Medical Systems) to evaluate the cardiac function at 28 days after MI; this procedure was repeated 3 times with the same equipment in the same examiner. Under electrocardiograph (ECG) monitoring of heart rate, 2D images of the hearts at the level of the greatest LV diameter were acquired in long-axis views. The LV ejection fraction (LVEF) was measured from 2D long-axis views of the infarcted area. Twenty-eight days after MI, the animals were euthanized to remove the hearts, and the infarct size was determined with Masson trichrome staining (Sigma). In brief, the hearts were simply trimmed to eliminate the upper part of the ligated area after immersion in 4% formaldehyde for 48 h. The remainder of the heart was cut into approximately 5-mm specimens perpendicular to the axis of the LAD coronary artery that were mounted on plastic and processed with paraffin embedding, producing 4-μm paraffin tissue sections. To measure the severity of myocardial fibrosis, the slices were stained with Masson trichrome to measure the average ratio of the fibrotic area to the entire LV cross-sectional area (fibrosis area %) using ImageJ software.

For the histological evaluation, kidney tissues were extracted, sectioned, and stained with standard protocols. Masson’s trichrome staining was performed with Masson Trichrome staining (Sigma-Aldrich, St. Louis, MO, USA) kits following the manufacturers’ instruction. The primary antibody used for immunostaining was anti-alpha smooth muscle actin (SMA) (NBP1-30894, Novus Biologicals, Littleton, CO, USA) immunohistochemical staining was performed with Envision DAB + kits (Dako, Basel, Switzerland) following the manufacturer’s instructions.

Five weeks after AMI, animals were euthanized to remove the hearts. Both Masson trichrome staining (Sigma) and Sirius Red staining were used for determining infarct size and fibrosis, and hematoxylin-eosin (HE) staining was performed to evaluate inflammatory cell infiltration. In brief, the hearts were simply trimmed to eliminate the upper part of ligation after immersion in 4% formaldehyde for 48 h. The remainder of the heart was cut at 1 mm below the ligation point perpendicular to the axis of the left anterior descending coronary artery and processed with paraffin embedding. To elucidate the severity of myocardial infarction, the slices with 4 μm of paraffin tissue were stained with Masson trichrome to measure the average ratio of the fibrotic area to the entire LV cross-sectional area (infarct area/LV%), and Sirius Red staining was used to calculate the average ratio of collagen area to entire LV area (collagen area/LV %), with the use of Image J software (Version 1.51j8, NIH) [25 (link)–27 (link)].

The 8 μm-thick transverse sections were used to determine the amount of collagen and muscle present in the bladder after Masson trichrome staining (Sigma-Aldrich, Switzerland). The muscle to collagen ratio in the bladder was quantified by imaging five randomly chosen areas per animal with a bright-field microscope (10x; Zeiss, Axio Scan.Z1). Images were imported in Fiji, the region covered by each channel was thresholded, and the area calculated in %.