Tcs sp8 confocal microscope

Hearts were frozen in OCT compound and cut to 6 μm longitudinal sections. Sections were thawed, fixed with 3.7% formaldehyde solution and were blocked with 10% mouse serum. Slides were treated with 0.1% Triton X-100 and incubated with primary antibody against α–smooth muscle actin (α-SMA; 1:200, rabbit IgG, ab5694, Abcam, Cambridge, UK) and discoidin domain-containing receptor 2 (DDR-2; 1:50, goat IgG, sc7555, Santa Cruz, Texas, USA) respectively for 1 hr at room temperature in PBS with 0.1% Triton-X100 and 10% mouse serum. Secondary antibody was Alexa Fluor-594 chicken-anti-rabbit IgG and Alexa Fluor-488 chicken-anti-goat IgG (Invitrogen) and nuclei were stained with DAPI. Confocal imaging was performed using a TCS SP8 confocal microscope (Leica Microsystems, Germany).

Titanium sheet and diamond-coated silicon wafers
were cut into
2.2 × 2.2 cm² squares. The diamond films were terminated
with hydrogen and oxygen as described in Section 2.1.3. The substrates were sterilized in 70%
ethanol for 10 min and air-dried inside a laminar flow hood. The substrates
were adhered to the bottom of a reusable eight-well silicon insert
(flexiPERM^R, Heraeus Instruments). Cells were seeded at
the densities stated in Section 2.4.1 and cultured for 5 days. The medium
was changed every second day, and on day 5, cells were fixed with
4% paraformaldehyde for 15 min. Cells were then permeabilized with
0.2% Triton X-100, blocked with 4% BSA/4% FBS, and incubated overnight
at 8 °C with mouse antivinculin antibody (clone hVIN-1, Sigma-Aldrich).
The antivinculin antibody was detected with goat antimouse antibody-AlexaFluor
488 (Thermo Fisher Scientific). Cells were counter-stained with DAPI
and Phalloidin-Atto 565 (Sigma-Aldrich). Finally, substrates containing
the stained cells were mounted on #1.5 glass coverslips using Mowiol^R (Sigma-Aldrich) mounting media. Specimens were imaged using
a TCS SP8 confocal microscope (Leica Microsystems) equipped with hybrid
detectors, white and blue diode lasers, and a 40× immersion objective
(NA = 1.1). Whole volume images of the cells were acquired with a z-step of 0.5 μm.

After cultured in 6-well plates under the different experimental conditions for 48 h, HK-2 cells were washed in PBS and incubated with MitoTracker Red CMXRos (Beyotime Biotechnology, Shanghai, China) using a working concentration of 200 nM at 37°C for 30 min. After the dye was removed by washing, the cells were immediately imaged using a TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany).

Confocal images were taken using the Leica TCS SP8 confocal microscope (Leica Microsystems, Wetzlar, Germany). For imaging of focal adhesions, cells were stained with paxillin antibody (Supplemental Table 1) and quantification was performed using ImageJ (see Supplemental Methods). F-actin fibres were stained with TRITC-phalloidin in Vectashield mounting medium (H-1600; Vectorlabs, Burlingame, CA, USA). KI67 staining (Supplemental Table 1) was performed under Boyden chamber assay conditions. KI67 positive and negative fractions among a total of 200 counted cells per condition and replicate (n = 3) was determined and normalized to the total number of counted cells.

Cells were fixed in phosphate buffered saline (PBS) containing 4% paraformaldehyde for 15 min at room temperature, washed with PBS, and treated with PBS containing 0.1% Triton X-100 and 5% goat or donkey serum for 30 min at room temperature. Thereafter, cells were stained with the following primary antibodies for 1 h at room temperature or overnight at 4°C: anti-NANOG (1:100, R&D Systems), anti-TRA-1-81 (1:100, Millipore), anti-cardiac troponin T (cTnT; 1:200, Thermo Scientific), and anti-dystrophin (1:100, MANDRA1, Sigma-Aldrich). Next, cells were stained with the following secondary antibodies for 1 h at room temperature: Cy3-conjugated donkey anti-mouse IgM (1:500, Jackson ImmunoResearch), Alexa Fluor 488-conjugated donkey anti-goat IgG (1:500, Invitrogen), Alexa Fluor 568-conjugated donkey anti-mouse IgG (1:500, Invitrogen) and a Zenon Alexa Fluor 488 labelling kit (Invitrogen). Nuclei were counterstained with 10 μg/mL Hoechst 33342 (Invitrogen). Images were acquired using a fluorescence microscope (BZ-X700 or BZ-X710, Keyence). Confocal images were captured by TCS SP8 confocal microscope (Leica Microsystems).

The ability of dendrimer/drug complexes to induce the apoptosis and necrosis in MCF7 and HEP G2 cells was evaluated by confocal microscopy using double fluorescent dye staining of orange acridine (OA) and ethidium bromide (EB). Both of these fluorescent dyes can stain cell nuclei after intercalation with DNA. While OA can be easily uptaken by cells and stains the nucleus green, EB is able to stain the nucleus of damaged cells only in red. Confocal analysis allows the indication of the fractions of the cells that are early and late apoptotic, necrotic or healthy. The cells were treated with dendriplexes for 24 h and stained with dual fluorescent staining solution (2 μL) containing 100 μg/mL AO and 100 μg/mL EB for 2 min and covered with a coverslip. Then, cells were washed with PBS and visualized using a Leica TCS SP8 confocal microscope (Wetzlar, Germany) with the objective 63×/1.40 (HC PL APO CS2, Leica Microsystems, Wetzlar, Germany).
The cells with normal morphology and a green nucleus were recognized as living cells, with green nucleus and condensed or fragmented chromatin as early apoptotic cells, with condensed or fragmented red chromatin as late apoptotic cells and with red nucleus as necrotic cells. Leica Application Suite X (LAS X, Leica Microsystems, Germany) software was used for the export of images.