Fv1000 clsm

RNA probes for rat IL-6 mRNA were prepared as previously described [26] (link). IL-6 mRNA in situ hybridization was performed in spinal cord sections of naïve and injured animals at 6 hours, 1, and 2 weeks after SCI. Briefly, sections were permeabilized in PBST (PBS, 0.3% Tween-20), followed by overnight hybridization at 45°C with 10 ng/μl of riboprobe in hybridization solution (4 mM Tris-HCl pH 7.5, Poly-A 0.5 mg/ml, salmon sperm 0.5 mg/ml, 50% formamide, 20% dextran sulphate, 0.1 M DTT, 0.3 M NaCl, 1X Denhardt’s solution, 1 mM EDTA pH 8.0). Next, sections were washed twice for 15 min in 2X SSC at 45°C, 4×15 min with 0.1X SSC at 55°C, once for 10 min at RT in 5X MAB (2.5 M maleic acid; 3.75 M NaCl; pH 7.5), and three times for 10 min at RT in 1X MABT (0.5 M maleic acid; 0.75 M NaCl; 0.1% Tween-20; pH 7.5). Sections were then incubated with blocking solution (1X MAB, 20% donkey serum, 2% blocking agent; 0.1% Tween-20) for 1 h at RT followed by overnight incubation at RT with anti-digoxigenin antibody conjugated to alkaline phosphatase (Roche) (1∶2000) in blocking solution. After washing 4×20 min in 1X MABT and 3×10 min in B3 (0.1 M Tris-HCl, pH 9.5; 0.1 M NaCl; 50 mM MgCl2; 0.1% Tween-20), sections were developed for 4 h with NBT/BCIP (Roche) (1∶50) in B3 solution, and images were acquired (Olympus FV1000 CLSM Olympus).

Flow cytometry was employed to determine the cellular uptake of PEG-CD/AD nanoparticles in HepG2 cells. Briefly, the cells were seeded on a 12-well plate and cultured for 24 h. Then the cultural media were replaced with fresh media containing free DOX or PEG-CD/AD nanoparticles at a DOX concentration of 2 μg/mL. After incubation for determined periods, the cells were collected and analyzed on a BD FACSVerse™ flow cytometer (BD Biosciences, USA).
For confocal laser scanning microscope (CLSM) study, the cells were placed onto 12-well glass plates. After incubation for 24 h, the cells were treated as the same as flow cytometry analysis. Then the cells were washed with PBS, fixed with 4% formaldehyde and the cell nuclei were stained by DAPI. Thereafter, the cells were observed under FV 1000 CLSM (Olympus, Japan).

Cultured cotyledons were loaded with a membrane-permeable Ca²⁺-sensitive dye, Oregon Green BAPTA-1 acetoxymethyl ester (OGB-1) at a concentration of 20 µM, for 3h at 4 °C. The loaded cotyledons were then incubated on liquid MS medium for 2h at 26 °C to allow cleavage of the Oregon Green ester by cytosolic esterases, hence trapping the membrane-impermeant Oregon Green dye in the cell cytosol (Zhang et al., 2015 (link)). Transverse hand-cut sections were then prepared and stained with 0.1% (w/v) tetrazolium blue (prepared in PBS buffer containing 100mM sucrose) for 20min to identify viable cells for microscope observations. Thereafter, the tissue sections were counterstained with 0.1% (w/v) Calcofluor White for 30 s to outline cell walls of adaxial epidermal cells, before being mounted in 200 µl of PBS buffer containing 100mM sucrose and visualized using an Olympus FV1000 CLSM. A 405nm UV laser with a 440–490nm emission filter set was used to visualize Calcofluor white fluorescence, while a 473nm laser with a 510–550nm emission filter set was used to visualize OGB-1 fluorescence.

Transmission electron microscope (TEM) images were acquired on a JEOL 2011 TEM (JEOL Ltd., Tokyo, Japan) with an accelerating voltage of 200 kV. UV–vis spectra were recorded on a PerkinElmer Lambda 750 spectrophotometer. Dynamic light scattering (DLS) was performed on a Nano ZS90 Zetasizer (Malvern Panalytical, Malvern, UK) to determine the hydrodynamic particle size of the samples. Confocal laser-scanning microscope (CLSM) images were obtained on a FV1000 CLSM (Olympus, Tokyo, Japan). Quantitative elemental analysis was performed on an inductively-coupled plasma optical emission spectrometer (ICP-OES, Agilent Technologies, Santa Clara, CA, USA). Flow cytometry (FCM) analysis for apoptosis assays was performed using a BD LSRFortessa flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). The western blot results were determined by Electro-Chemi-Luminescence display devices (Thermo Fisher Scientific, San Jose, CA, USA).

Biofilm architecture and thickness were acquired using an FV1000 CLSM (Olympus, Tokyo, Japan) equipped with a 60 × 1.35NA lens. The excitation wavelength for green-live SYTO 9 dye was 488 nm and emission wavelengths of 515 nm were collected. For the red-dead PI stain, the excitation was 530 nm and emission was recorded at 617 nm. Three-dimensional projections of biofilm structures were reconstructed with quantitative structural parameters of the biofilms using the Easy 3D function of the IMARIS software (Bitplane AG, Zürich, Switzerland).