4 6 diamidino 2 phenylindole (dapi)

MOVAS-1 cells were grown on glass coverslips and calcified as described above. After incubation with Fluorescein-BP probe 1 (1 μM) for 2 hours, the media was changed, the monolayer was washed twice with HBSS and fresh HBSS containing 500 nM CellMask Orange Plasma Membrane Stain (Thermo Fisher) was added for 10 minutes. The monolayer was washed with phosphate buffered saline (PBS, 2×) and water (1×), fixed with 10% neutral buffered formalin (NBF) for 15 minutes and washed with PBS (2×) and water (1×). The cell monolayers were permeabilised with Triton X-100 (0.05%, 3×) for 5 minutes and incubated with DAPI (300 nM; Life Technologies) for 5 minutes. Excess DAPI was removed by washing with water (1×). Glass coverslips were mounted onto slides with Prolong Gold Anti-Fade Reagent (Life Technologies). Fluorescence signal was detected under a Zeiss Axiovert 25 inverted microscope and/or Zeiss Confocal LSM 710 at 488 nm (Fluorescein-BP), 554 nm (CellMask Orange) and 350 nm (DAPI).

Embryos were chilled on ice in E3 medium for 15 min, followed by incorporation of 10 mM BrdU (Sigma-Aldrich; Missouri, USA) for 20 min on ice. Afterwards embryos were washed several times with pre-warmed E3 medium and were finally fixed with 4% PFA in PBS overnight at 4°C. Fixed embryos were stored in methanol at −20°C overnight. Permeabilization was performed by ProteinaseK treatment (10 μg/ml) for 10 min at room temperature, followed by a PBST wash step. Samples were treated with 2 N HCl for 1 h at 37°C. Finally, embryos were blocked in 1% BSA and incubated overnight with anti-BrdU (mouse; 1:100; abcam; Cambridge, GB) and anti-GFP (rabbit; 1:1,000; abcam; Cambridge, GB) at 4°C. On the next day embryos were washed with PBST and subsequently stained with secondary antibody [Alexa488; 1:1,000; (Molecular Probes; Oregon, USA), cy3; 1:1,000; (Jackson Immunoresearch; Pennsylvania, USA)] and DAPI (1:1,000; Carl Roth; Karlsruhe, Germany). Mounting was performed in Mowiol. For quantification BrdU-DAPI-positive cells within the primordium of all embryos were counted at a ZEISS LSM510.

The Caco-2 cells, L929 cells, and M1Φ were incubated with the coumarin 6-labeled PLGA NPs or CS-PLGA NPs for 1 h, respectively. The cells were harvested and fixed with 4% paraformaldehyde for 15 min and stained with DAPI for fluorescence imaging (Carl Zeiss, Oberkochen, Germany). The harvested cells were also analyzed by flow cytometry (ACEA NovoCyte 3000, Agilent, USA) for intracellular uptake efficiency.

Cross sections (approximately 5–10 mm thick) of the internodes of PdRanBP transgenic hybrid poplars and WT stems, as well as the tips of EGFP-PdRanBP transgenic hybrid poplars and WT stems, were fixed overnight at room temperature (RT, 22 °C) in a formalin–alcohol–acetic acid (FAA). The samples were then embedded in paraffin wax, cut into 8-μm sections using a microtome (Leitz, Wetzlar, Germany), and dehydrated through an alcohol series. WT stems and cross sections of the internodes of PdRanBP transgenic hybrid poplars were stained with toluidine blue O (TBO), as described by Abbott et al. [66 (link)]. WT stems and cross sections of the tips of EGFP-PdRanBP transgenic hybrid poplars were briefly stained with DAPI (1 mg/mL in mounting medium [Vectashield; Vector Labs, Burlingame, CA, USA]), as described by Jasencakova et al. [67 (link)].
The number of radial cell layers and the overall widths of the xylem, phloem and cambium region of PdRanBP transgenic hybrid poplar were measured using an inverted fluorescence microscope. The number of nuclei was determined by counterstaining with DAPI (Carl Zeiss). The images were obtained using a digital camera system (AP-1; Apogee Instruments Inc., Tucson, AZ, USA).

Live cells were spotted on 1% agarose pads (prepared with phosphate-buffered saline [PBS]) between a glass slide and a coverslip. When indicated, cells were stained with 5 µg/ml DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich) and 5 µg/ml FM4-64 (Life Technologies) just before imaging. Image acquisition, analysis, and processing were performed as described previously (45 (link)) using filter sets 31, 49, and 46 (Carl Zeiss) to image FM4-64, DAPI, and GFPmut2-associated fluorescence, respectively. We used a parameter set modified from algorithm 1 of the MicrobeTracker suite (46 (link)) to obtain subpixel cell outlines. Quantitative analysis and plots from the MicrobeTracker data were done on MATLAB (MathWorks, Inc.) using homemade scripts. The values of cell width were obtained by dividing the cell area by the cell length.

Human primary neurons grown on 50 μg/ml poly-D-lysine (Sigma Aldrich)-coated 35-mm glass coverslips were exposed to Tat or TNFα for 48 h. After treatment, the neurons were fixed in PBS containing 4 (w/v) paraformaldehyde for 20 min at room temperature, permeabilized with 0.5% (v/v) Triton X-100 for 15 min at room temperature and then blocked in PBS containing 3% bovine serum albumin for 1 h. The cells were then incubated with anti-PINCH antibody (1:200 dilution; BD Biosciences) overnight at 4 °C. After 3 washes with 1× PBS, the neurons were incubated for 1 h with Alexa Fluor 488-conjugated anti-mouse IgG (Invitrogen; 1:200 dilution) and rhodamine phalloidin (1:1000, Invitrogen) to simultaneously label PINCH and actin, respectively. After 1-h incubation, neurons were washed with PBS and mounted with Vectashield containing DAPI (Vector Lab., Burlingame, CA, USA). Confocal images were obtained at 405 nm (DAPI), 488 nm (PINCH), and 561 nm (actin) excitations respectively using a 63× oil objective (LSM 800; Carl Zeiss, Inc.). The length of actin filaments was manually measured with ImageJ software (NIH). All of the images taken had a resolution of 0.592 pixels/μm. Using measure analysis tool in ImageJ we measured the pixel length of each filament in an image.

TUNEL assay was performed to evaluate BMEC apoptosis in the brain of SAP rats using a TUNEL staining kit (POD, Roche, USA). After dewaxing, rehydration and inactivation of endogenous peroxidase, the sections were incubated with TUNEL reaction mixture at 37 °C for 60 min. The nuclei were stained with DAPI (1:1000, Sigma). TUNEL-positive and DAPI-positive cells were counted under a fluorescence microscope (Carl Zeiss).