4 6 diamidino 2 phenylindole dapi

To immunostain brain slices, adult mouse brains were infused with Tissue-Tek (Sakura Finetek, Tokyo, Japan), frozen in liquid nitrogen and sectioned at 10 µm using a cryostat (HM500-OM, Carl Zeiss, Oberkochen, Germany). The sections were mounted on silane-coated coverslips and dried for ~30 min at room temperature. The samples were fixed with 3.7% formaldehyde in phosphate-buffered saline (PBS; 137 mM NaCl, 8.1 mM Na₂HPO₄, 1.5 mM KH₂PO₄, and 2.7 mM KCl, pH 7.4) for 10 min at room temperature and permeabilized with 0.5% Triton X-100 in PBS. After blocking with 10% FCS in PBS, the samples were incubated with the anti-RNG105 antibody over night at 4°C. After washing with PBS, the samples were incubated with Alexa488-conjugated anti-rabbit IgG (1:400, Jackson ImmunoResearch, West Grove, PA, USA) and 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI) (Wako Pure Chemical Industries) for 1 hr at room temperature to label RNG105 and nuclei, respectively. To immunostain cultured neurons, neurons at 12 DIV were fixed and stained in the same way. Fluorescence images were acquired using an A1 confocal laser microscope equipped with a Ti-E inverted microscope (Nikon, Tokyo, Japan) with a 10 × objective lens or a PlanApo VC60 × oil objective lens.

For immunostaining, the ESCs were fixed in 10% formaldehyde, permeabilized with 0.1% Triton X-100, blocked with 1% BSA, and then stained with an anti-Oct3/4 antibody (rabbit polyclonal IgG 1:100; Santa Cruz Biotechnology, Dallas, TX) or an anti-FGF5 antibody (rabbit polyclonal IgG 1:100; Santa Cruz Biotechnology) as primary antibodies. Primary antibody binding was visualized using AlexaFluor 546-conjugated anti-rabbit polyclonal IgG (1:2,000; Invitrogen, Carlsbad, CA). Nuclei were stained with 0.4 μM 4′,6-diamidino-2-phenylindole (DAPI; Wako Pure Chemical Industries, Osaka, Japan). Micrographs were obtained using a BZ-8100 microscope (Keyence, Osaka, Japan).
For flow cytometry analysis, all cells were removed from culture dishes using 0.02% (w/v) EDTA-4Na in PBS, and then 0.1 μg/mL propidium iodide (PI; Wako Pure Chemical Industries) was added to aid identification of dead cells. A JSAN flow cytometer (Bay Bioscience Co., Kobe, Japan) was used for data acquisition.

The retinal slices were incubated with 10 mM citrate buffer (pH 6.0) for 5 min at 121°C for antigen retrieval. After washing, the slices were blocked and incubated with primary antibodies (Table S3) overnight at 4°C. After another washing step, the slices were incubated with the appropriate Alexa Fluor 488- or 594-conjugated secondary antibody (1∶500; Life Technologies) for 1 hour at 25°C, followed by counter-staining with 2 µg/mL 4′,6-diamidino-2-phenylindole (DAPI; Wako Pure Chemical Industries, Osaka, Japan). Immunoreactivity signals were photographed using a fluorescence microscope (VB-7000 or BZ-9000, Keyence, Osaka, Japan).

Reagents included De Man Rogosa and Sharpe (MRS) media (Becton Dickinson Difco, U.S.A.), hipolypeptone (Nihon Seiyaku, Japan), beef extract (MP Biomedicals, LLC, France), yeast extract (Becton Dickinson Difco), TBO (Waldeck, Munster), and Percoll (GE Healthcare Life Sciences, Japan). Glucose, Tween 80, K₂HPO₄, sodium ascorbate, L-cysteine-HCl, NaNO₃, MgSO₄, KH₂PO₄, NaH₂PO₄, NaCl, and 4′,6-diamidino-2-phenylindole (DAPI) were procured from Fujifilm Wako Pure Chemical Corporation, Japan. Data were collected using a spectrophotometer (UV-1200, Shimadzu, Japan) and a fluorescence microscope DMRXA/RD (Leica Microsystems, Germany). Cultures were prepared using fixed-type (model of rotor: BN 4–6) (H-201FR, Kokusan, Japan) and swing-type (model of rotor: RF 110) centrifuges (H-500FR, Kokusan). Deionised and doubly distilled water was used in all experiments.

The cells were fixed with 4% paraformaldehyde at 4 °C for 5 min and permeabilized with 0.1% Triton X-100 at room temperature for 20 min in the presence of a protein-blocking solution consisting of PBS supplemented with 5% normal goat serum (Agilent Technologies, Inc., Santa Clara, CA, USA). The cells were incubated overnight with primary antibodies in PBS at 4 °C. They were washed extensively in PBS and incubated at room temperature for 30 min with secondary antibody. The nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; diluted 1:500; FUJIFILM Wako Pure Chemical) in PBS at room temperature for 30 min. To prevent fading during microscopy, the cells were mounted in DakoCytomation Fluorescent Mounting Medium (Agilent Technologies, Inc.). Immunofluorescence images were visualized and recorded using a Biorevo BZ-9000 fluorescence microscope (Keyence Corporation, Osaka, Japan).

For observing whether EPC-MVs could merge with H9c2 CMs, a lipid membrane-intercalating fluorescent dye (PKH26) was used to label EPC-MVs before co-incubation. Briefly, 50 µg/ml EPC-MVs was mixed with 2 ml of PKH26 (2×10⁻⁶ M; Sigma-Aldrich, St. Louis, MO) at room temperature (RT) for 5 min. The labeled mixtures were dialyzed in 2 ml of 1% bovine serum albumin (BSA) and ultracentrifuged at 100,000 g for 60 min at 4°C to pellet the labeled MVs. After washed with EBM-2, the pellet was suspended with 1 ml of culture medium and added into H9c2 cells for 24 h incubation. The 4′, 6-diamidino-2 -phenylindole (DAPI, 1 ug/ml; Wako Pure Chemical Industries Ltd) was used for nuclear staining. Cell images were taken using an inverted microscope (EVOS, NY).