Tcs sp8 inverted confocal microscope

Transmission electron microscope (TEM) images were obtained using an H-800 microscope (Hitachi, Japan). Zeta potentials were measured on a Malvern Zetasizer Nano-ZS. PXRD experiments were recorded on a D/Max-2500 X-ray diffractometer using Cu-Kα radiation (λ = 1.5418 Å). X-ray photoelectron spectra (XPS) were recorded using a Thermo Fisher Scientific ESCALAB 250Xi spectrometer (USA). The Ca²⁺ and Zr⁴⁺ ion contents were measured by inductively coupled plasma mass spectrometry (ICP-MS, 8900, Agilent, USA). The UV-vis spectra were recorded on a Cary 60 UV-vis spectrophotometer (Agilent technologies). The fluorescence measurements were recorded on an F-4600 (Hitachi, Japan) instrument. CCK-8 assay was performed on a microplate reader (DG5033A, China). Confocal fluorescence imaging was performed using a Leica TCS SP8 inverted confocal microscope (Leica, Germany). The cell images were acquired using a 20× objective. In vivo imaging was performed using an IVIS Lumina LT imaging system.

Cells were plated on glass coverslips coated with collagen I in 24 well plates and grown to 3 to 5 days postconfluency. In Leiden, cells were fixed with 4% paraformaldehyde (PFA; Alfa Aesar) without washing, then rinsed with PBS and blocked and permeabilized for 20 minutes in blocking buffer (PBS; 5% Normal Goat Serum [Dako]; 0.02% saponin [Sigma‐Aldrich]). In Kingston, cells were washed once with PBS and fixed with Fixation Solution (BD Bioscience). Cells were rinsed with PBS and permeabilized with PBS/0.1% Triton x‐100 for 10 minutes on ice, then washed with PBS and blocked for 20 minutes with Protein Block (Dako).
After fixation, permeabilization, and blocking, cells were stained with primary antibodies for VWF and VE‐cadherin (Table S1 in supporting information) diluted in either blocking buffer or PBS/1% bovine serum albumin (BSA) (Sigma‐Aldrich). Nuclear staining was performed with either Hoechst (Thermo Fisher Scientific) or DAPI (Sigma‐Aldrich) diluted in PBS. Coverslips were mounted by Mounting Media (Dako) or ProLong Diamond Antifade Mountant (Thermo Fisher Scientific) and cells were imaged using the Leica TCS SP8 inverted confocal microscope (Leica Microsystems) with the white light laser (WLL), hybrid detectors (HyD), and the HC PL APO CS2 63x/1.40 oil immersion objective. Images were acquired and analyzed using the LAS‐X Software (Leica Microsystems).

HEK293 cells (0.4 × 10⁵) seeded onto Lab-Tek (Nunc) were transfected with mutated or unmutated pCMV-iRFP-2A-SPI1. After 48 h, cells were washed with PBS, fixed for 15 min in 4% paraformaldehyde, and permeabilized for 15 min in 0.1% Triton X-100 in PBS at room temperature. After 30 min in blocking buffer (PBS 0.1% Triton X-100 with 2% BSA), cells were incubated for 2 h with a rabbit anti-PU.1 monoclonal antibody (EPR3158Y; Abcam), washed again, and incubated for 1 h in blocking buffer containing goat anti-rabbit Alexa Fluor 488 antibody (Invitrogen). After a final wash, cells were DAPI stained and mounted on slides using Fluorescence Mounting Medium (Dako). Images were obtained on a Leica TCS SP8 inverted confocal microscope with a 63× oil immersion objective (Leica Microsystems). Images were analyzed with ImageJ software.

Samples were characterized by Ultra high resolution field emission scanning electron microscopy (fe-sem, Nova NanoSEM 450) and transmission electron microscopy (TEM, Tecnai G2 F30) at an accelerating voltage of 200 kV. X-ray diffraction (XRD) patterns of the as prepared samples were recorded on a SHIMADZU XRD-7000S diffractometer with Cu K as the radiation source in the a range of 10-90° with a scan speed of 5° min⁻¹. The Brunauer–Emmett–Teller (BET) specific surface areas were measured by JW-BK132F at −196 °C. The pore volumes and pore diameter distributions were calculated with the Barrett–Joyner–Halenda (BJH) model. Particle sizes were acquired on a Malvern Zeta sizer Nano ZS. X-ray irradiation was performed using BJI-1 (Xianwei, Shanghai, China) at a voltage of 60–75 kV and a current of 0.15–0.35 mA. Confocal laser scanning microscopy (CLSM) images were recorded on a Leica TCS SP8 inverted confocal microscope. DOX loading and release studies were carried out in a Shimadzu UV-1800 spectrophotometer. The determination of ROS was measured using Hitachi F-7000 fluorescence spectrophotometer. The cell cytotoxicity was assessed by BioTek SynergyH1 Full-featured microplate detector.