Ab77232

Myricetin (DY0103, HPLC ≥98%) was purchased from MUST Biotechnology Co., Ltd. (Chengdu, China) for animal study. For cell experiments, Myricetin (70050) and DMSO (D2650) was obtained from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle medium (DMEM) and horse serum (16050130) were bought from Gibco (Carlsbad, CA). Fetal bovine serum (FBS) was purchased from Hyclone Laboratories, Inc. (Logan, UT, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo (Kumamoto, Japan). mirVana™ miRNA Inhibitors (rno-miR-499-5p, MH11352), mirVanaTM miRNA mimics (rno-miR-499-5p, MC11352), Lipofectamine RNAiMAX Transfection Reagent (13778083) and antibody against PGC-1α (PA5–38021) were bought from Invitrogen (Massachusetts, USA). Antibodies against slow skeletal myosin heavy chain (ab11083), fast skeletal myosin heavy chain (ab91506), Sox6 (ab30455), tnni1 (ab231720) and myoglobin (ab77232) were purchased from Abcam (Cambridge, UK). Antibody against Cytochrome C (Cyt C, sc-13,561) and β-actin (sc-47778) were purchased from Santa Cruz Biotechnology (CA, USA).

After all MRI studies, mice were sacrificed and hearts were extracted. Heart samples were thoroughly washed in saline solution and embedded in optimal cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA, USA) and cut into 5 μm thick short-axis slices. To evaluate the necrotic myocardium, immunohistochemistry was performed on frozen tissue sections using a primary antibody against myoglobin (ab77232, Abcam, Cambridge, MA, USA) [24 (link), 25 (link)]. To detect necrotic myocardium, a HistoMouse™-MAX kit (Invitrogen, Waltham, MA, USA) was used to immunostain the sections, and color was developed using the Fast DAB with metal enhancer tablet set (Sigma-Aldrich). To analyze macrophage distribution, heart sections were stained with a primary antibody against CD68 (ab53444, Abcam) and Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes, Eugene, OR, USA). The sections were counterstained with DAPI to visualize the nuclei. We evaluated colocalization of MNPs and macrophages in inflamed myocardium by color-coding the confocal laser scanning microscopic images using red for RITC (MNP), green for Alexa Fluor488 (macrophages), and blue for DAPI (nuclei). Stained tissue sections were digitally scanned using a slide scanner (SCN400, Leica, Oberkochen, Germany).

MSCs were suspended to a final concentration of 1.0 × 10⁶ cells/ml in phosphate-buffered saline (PBS) containing 0.1% (w/v) NaN₃ and 5% (v/v) FBS. For specific staining, 100 μl of each MSCs suspension was incubated with 10 μl of each primary antibody for 20 min at 4 °C. The following primary antibodies were used: mouse monoclonal phycoerythrin (PE)-conjugated anti-CD73, anti-CD105, anti-CD90, anti-CD45 (BD Biosciences) and rabbit monoclonal anti-myoglobin (ab77232) from Abcam (1:50). Nonimmune, isotype-matched and isotype-conjugated mouse IgG1k and goat anti-rabbit IgG fluorescein isothiocyanate (FITC)-labeled (ab6717) antibodies were used as negative controls. For intracellular myoglobin staining, MSCs were permeabilized with Fix & Perm Kit (Life Technologies, Carlsbad, CA, USA), according to the manufacturer’s instructions, before incubation with anti-myoglobin antibody; FITC-conjugated anti-rabbit IgG was used as secondary Ab for Mb detection. Cells were assessed with Navios flow cytometer (Beckman Coulter, Indianapolis, USA); the emitted fluorescent signal of 10,000 events for each sample was acquired and analyzed using the Kaluza Analysis software (version 1.3, Beckman Coulter, Brea, CA, USA) as described [12 (link)].