Hoechst 33258

Cell apoptosis assay was assessed by using PE Annexin V Apoptosis Detection Kit I (BD Biosciences, San Diego, CA, USA) and Hoechst 33258 staining as previously described [18]. Briefly, following different treatments, MC3T3-E1 cells were collected and resuspended in 100 μL binding buffer, stained with Annexin V-PE and 7-AAD at room temperature in the dark for 15 min. Stained cells were analysed using a FACSCantoⅡ flow cytometer (BD Biosciences, USA). For pathway blocking experiments, cells were pre-incubated with PD98059 (10 μM) for 2 h, then cells were treated with EPC-MVs or EPC-MVs-miR126. Cell apoptosis rate was quantified in three times independent experiments.
For Hoechst 33258 staining, cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min after EPC-MVs or EPC-MVs-miR126 treatment for 48 h, then stained with Hoechst 33258 (Sigma, Louis, MO, USA) according to the manufacturer’s instruction. Morphologic changes were observed under a fluorescence microscopy in four independent fields.

Cell death was assessed using propidium iodide (PI; Sigma) staining in combination with Hoechst 33258. Briefly, PI was added to cultures at 200 μg/ml in cell culture medium and left to incubate for 15 min at 37 °C. The medium was then removed and the cultures were rinsed in PBS before fixing in 4% paraformaldehyde (PFA) at 4 °C. Gels were incubated with Hoechst 33258 (1 μg/ml; Sigma) in PBS for 10 min, before 3 × 5 min washes in PBS. Fluorescence microscopy was used to determine cell viability. Images were captured using a Zeiss Axiolab A1 fluoroscence microscope and Zeiss AxioCam C1. Three fields were randomly selected per gel. The % of dead cells for each cell population was determined by counting the number of PI stained cells and the total number of cells, as determined by Hoechst staining. For neurons, the number of βIII-tubulin immunopositive cells was calculated as a percentage of the total number of cells/field and compared to the number of PI stained cells to determine cell death.

H/R PC12 cells treated with NC or miR-375 mimic were cultured in six-well plates, and Hoechst 33258 (Sigma–Aldrich, St. Louis, MO, U.S.A.) was added to the culture medium and incubated for 10 min, cells were then washed with PBS three times, and the counts of Hoechst-positive nuclei were detected by inverted fluorescence microscope using a 365-nm filter for Hoechst 33258.

MDA-MB-231 cells were grown in 12-well plates and incubated for 48 h with and without RSF EtOAc extract at IC₅₀. Morphological changes characteristic of apoptotic cells were observed using phase contrast inverted microscopy (MC-170 HD camera, Leica, Wetzlar, Germany) at 200× magnification. For Hoechst 33,258 staining, cells were treated then washed twice with PBS at room temperature, fixed with 4% paraformaldehyde, permeabilized using cold methanol, and stained with Hoechst 33,258 (Sigma) diluted in PBS (final concentration 0.5 µg/mL) for 30 min in the dark. Cells were examined for nuclear changes (i.e., chromatin condensation and nuclear fragmentation) using a fluorescence microscope attached to an Axiocam 506 color camera (Zeiss, Wetzlar, Germany).
For acridine orange/ethidium bromide dual staining, MDA-MB-231 cells were treated at IC₅₀ for 48 h, directly stained with AO/EB (4 μg/mL) for 5 min, and imaged immediately using fluorescence microscopy (EVOS, Carlsbad, CA, USA).

Cells were grown on coverslips in 6-well plate. The morphology and apoptosis of cells were examined by staining with cell-permeable DNA dye Hoechst 33258 (Sigma). Briefly, cells were fixed with ice-cold 70% ethanol in PBS for 30 min after exposure to TPL (25 nM) and/or X-ray (6 Gy) for 48 h, then stained with 5 μg/mL cell-permeable DNA dye Hoechst 33258 (Sigma) dissolved in Hanks' buffer for 10 min in the dark. Cells with homogeneously stained nuclei were considered to be viable, whereas the presence of chromatin condensation and/or fragmentation was indicative of apoptosis. To calculate the percentage of apoptotic cells, at least 200 cells in three different microscopic fields were evaluated.

For desmin immunostaining, myotubes were fixed with 4% paraformaldehyde on the 5th day of differentiation, and after 5-min permeabilization with 0.1% Triton X-100 in PBS, the samples were blocked in 0.1% bovine serum albumin (BSA) in PBS. For staining the differentiated myotubes, the samples were incubated overnight with mouse anti-desmin (Biocare Medical, 901-036-081214, Pacheco, CA, USA) primary antibody at 4 °C, followed by incubation with anti-mouse Alexa Fluor 488-conjugated secondary antibody (Jackson Immunoresearch, West Baltimore Pike, West Grove, PA, USA) for 20 min. Nuclei were stained with Hoechst 33258 (Sigma-Aldrich), and samples were coated with a fluorescent mounting medium (DAKO).
For visualization of actin filaments, the myotubes were fixed with 4% paraformaldehyde and incubated with PBS containing 0.9% Triton X-100 and 4% BSA for 30 min. Then, the samples were labeled with Alexa-647-conjugated phalloidin (Cell Signaling, #8878S). Following nuclear staining with Hoechst 33258 (Sigma-Aldrich), the samples were immediately processed for dSTORM and confocal imaging.

Mouse embryos were fixed in 4% paraformaldehyde (PFA) for 3–6h. Tissues were subsequently washed, dehydrated, embedded in paraffin wax, and sectioned at 3 μm. For immunofluorescence, a standard immunodetection protocol was followed as described in23 (link). Briefly, tissues were rehydrated and, when required, subjected to heat-mediated antigen retrieval in citrate buffer. After a blocking step in 5% donkey serum/ 0.2% Triton X-100, tissue sections were incubated overnight with primary antibodies and then for 1 h with secondary antibodies (Supplementary Table S3). Nuclei were stained with Hoechst 33258 (Sigma). Fluorescent images were captured using a Leica DMI 6000B widefield microscope or a Leica TCS SPE confocal microscope. For morphometrical analysis, total pancreas was sectioned at 3 μm and distributed as serial sections onto sets of 5 slides. At least 10 sections 45 μm apart per animal were analyzed using Image J software (http://rsb.info.nih.gov/ij/index.html).
INS1E cells were fixed in 4% PFA for 20 min at room temperature and permeabilized in PBS with 0.2% Triton X-100. After a blocking step of 1 h in 3% normal donkey serum, cells were incubated overnight with the indicated primary antibodies. After washes, cells were incubated with the secondary antibodies for 1 h at RT and. nuclei were stained for 3 min in a 1:500 dilution of Hoechst 33258 (Sigma).