Bx51 dot slide

For analysis of target protein expression patterns in rat hearts under pathological conditions, perfused hearts were fixed with 4% formaldehyde and embedded in paraffin. Three-micron-thick sections were mounted on gelatin-coated glass slides. After deparaffinization and washing with PBS, sections were incubated in blocking solution containing 2% normal horse serum, 1% BSA, 0.1% Triton X-100, and 0.05% Tween 20 in PBS. Slides were incubated with a mixture of primary antibodies (RIP3/p-MLKL or BNIP3/CAS3) at the appropriate dilutions in PBS containing 1% BSA for 24 h at 4°C, and fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG and rhodamine-conjugated goat anti-mouse were used as secondary antibodies (1:500). The nuclei were stained with DAPI and examined under virtual microscopy (BX51 Dot Slide; Olympus, Tokyo, Japan).

Five micrometer-thick longitudinal sections were cut from the apex to the base and mounted on a glass slide. Masson’s trichrome staining was performed according to standard protocols, and the fibrotic area was measured using ImageJ software. The terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL Assay Kit—BrdU-Red (Abcam, #ab66110) was used to determine the number of apoptotic and necrotic cells in the infarcted myocardium, as per the manufacturer’s instructions. TUNEL assay images were blindly captured and counted using a virtual microscope (BX51 Dot Slide; Olympus, Tokyo, Japan). To assess the relationship between the transplanted EV and ischemic myocardium, immunohistochemistry was performed. Sections were deparaffinized and incubated in 1% H₂O₂ to quench endogenous peroxidase. Cardiac tissues were blocked for 1 h with a mixture of 1% (w/v) BSA and 5% (v/v) horse serum and incubated with VE cadherin antibody (Abcam, UK, #ab205336) or α-SMA (Santa Cruz Biotechnology, #sc53015) at a ratio of 1:200. After washing thrice with PBS, the sections were incubated with secondary antibodies (Alexa Fluor 488, #ab150077; Alexa Fluor 594, #ab150116; at a ratio of 1:250, Abcam) for 1 h at room temperature, mounted with DAPI, and viewed under a confocal microscope (LSM 700, Carl Zeiss, Oberkochen, Germany).

To measure myocardial infarct size, 2–3 mL 2% Evans blue solution (Sigma, St. Louis, MO, USA) was transcardially perfused. The heart was subsequently removed and washed with saline. Prior to being divided into six 2- to 3-mm sections, the hearts were perfused with 1% TTC (Sigma, St. Louis, MO, USA) for 1 h at 37°C and incubated in 4% formaldehyde overnight at 4°C. The heart sections were photographed with a digital camera. The area at risk (red staining) indicating the ischemic area, the infarcted area (white staining), and the non-ischemic area (blue staining) were observed. The infarcted area was measured directly by planimetry of normal and infarcted left ventricular myocardia using ImageJ software. In infarcted hearts, apoptotic and necrotic cells were determined using PI and TUNEL staining. All PI-positive cells indicated necrotic cell death, and cells that were TUNEL positive only indicated apoptotic cell death. In brief, sections of heart tissue were incubated with PI without permeabilization, and then TUNEL staining was performed per the manufacturer’s instructions. The nuclei were stained with DAPI and examined under virtual microscopy (BX51 Dot Slide; Olympus, Tokyo, Japan).

A terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) assay was performed according to the manufacturer’s instructions (Millipore). hMSCs were plated in a four-well culture dish (1 × 10⁴ cells per well) and treated with 500 μM H₂O₂ for 6 hours with or without prior let-7b transfection. After the slides were rinsed with PBS, the cells were fixed with 10 % formaldehyde (Sigma) for 10 minutes. The slides were treated with 3.0 % H₂O₂ and TdT enzyme for 1 hour followed by digoxygenin-conjugated nucleotide substrate at 37 °C for 30 minutes. Nuclei were stained with 3,3′-diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA, USA) for 5 minutes, and the slides were counterstained with methyl green (Sigma). Dark-brown-stained nuclei indicated apoptotic cells. The slides were observed by a virtual microscopy (BX51/dot Slide; Olympus, Tokyo, Japan).