Lsm 710 confocal laser scanning microscope

Confocal fluorescence microscopy was used to investigate the cellular uptake and internalization of carriers. Raw 264.7 cells were grown in an 8-well μ-slide (Ibidi, Martinsried, Germany) until 50–60% confluence was achieved. Nuclei were stained by incubating the cells with DAPI. FITC-labeled mcDNA was encapsulated into R8-mannose/PEI particles and real live transfection was visualized using an LSM 710 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany) under 63× magnification and analyzed with the Zeiss LSM 710 laser scanning confocal microscope (Carl Zeiss SMT, Inc., Oberkochen, Germany). During the experiment, Raw 264.7 cells were maintained at 37 °C with 5% CO₂.

The intracellular ROS levels were examined using DCFH‐DA (Life Technologies‐Thermo Fisher Scientific) before visualization with a LSM710 Laser Scanning Confocal Microscope (Carl Zeiss) to quantify the fluorescence signals of the oxidized product (2′,7′‐dichlorofluorescein, DCF).
The Griess assay (Beyotime) was used to evaluate the amount of NO in the culture supernatant by measuring the concentration of nitrite (a stable NO breakdown product). An NO⁻ sensitive fluorescence probe DAF‐FM DA (Sigma) was used to detect intracellular NO.22 DAF‐FM DA (10 μmol/L) was used to label the cells at 37°C for 30 minutes before they were washed thrice with PBS. Fluorescence was detected using a LSM710 Laser Scanning Confocal Microscope (scale bars, 100 μm) (Carl Zeiss).

Time-lapse images for zebrafish circulation, the LTB₄ bath and the flow adhesion assay were obtained with an AXIO Zoom V16 microscope (Zeiss, Thornwood, NY, USA). Time-lapse fluorescence images for zebrafish neutrophil motility were acquired using a LSM 710 laser scanning confocal microscope (Zeiss, Thornwood, NY, USA) with a 1.0/20× objective lens at 1 min interval of 30 min. Neutrophils were tracked using ImageJ with the MTrackJ plugin and the velocity was plotted in Prism 6.0 (GraphPad, San Diego, CA, USA). Time-lapse fluorescence images for dHL-60 migration were acquired using a LSM 710 laser scanning confocal microscope (Zeiss, Thornwood, NY, USA) with a 1.0/20× objective lens at 10 s interval for 5 min. Cells were stained with 1 µM ER-tracker and 20 nM TMRM for 20 min, washed twice with Hanks' balanced salt solution (HBSS) and added to fibrinogen coated wells. After 30 min, cells were treated with 1 nM fMLP to induce chemokinesis.

Retinal flat mounts immersing in marker solutions were processed to visualize the vascular basement membrane. Prior to immersion staining, one-fourth of the retinal tissue in each sample was incubated for 45 mins at room temperature in 5% normal bovine serum in PBS containing 0.5% Triton-X-100 (0.5% T-PBS). Subsequently, the flat mounts were incubated overnight at 4°C in a marker solution containing rabbit polyclonal anti-type IV collagen antibody solution (1 : 300; ab19808; Abcam, Cambridge, UK) for basement membrane [9 (link)]. Fluorescent goat anti-rabbit immunoglobulin (Ig) G (1 : 45; BA1105; Wuhan Boster Biological Technology, Ltd., Wuhan, China) was treated as a secondary antibody. Subsequent to secondary incubation at 20°C for 5 mins, the retinal flat mounts were washed three times in 0.5% T-PBS, kept into DAPI for 5 mins, and washed another three times in 0.5% T-PBS. Then, the retinal flat mounts were prepared in a Vectashield (Wuhan Boster Biological Technology, Ltd.) and analyzed using a Zeiss LSM 710 confocal laser scanning microscope to determine the area and number of retinal neurocytes and the number of type-IV collagen strands.

Fixed cells
were stained for β₃-tubulin (1:500) and GFAP (1:1000)
using secondary antibodies tagged with fluorescent markers (Alexa
Fluor 488 and 555, 1:300); all antibodies and markers were obtained
from Abcam. Samples were mounted in DAPI-containing mounting media
(Vector Labs, Peterborough). Samples were imaged on a Nikon epifluorescence
microscope fitted with an auto-x-y scanning stage, allowing large-area
stitched image acquisition. All images were taken at ×20 magnification.
Confocal imaging was carried out on a Zeiss LSM 710 confocal laser
scanning microscope fitted with 20× objective. Layer slices were
captured at a resolution of 45 μm with successive layers being
pictured.

For immunostaining, cells were seeded onto eight-well Nunc Lab-Tek II Chambered Coverglass (ThermoFisher Scientific, Waltham, MA, USA, #155411), fixed, and permeabilized with 4% PFA (paraformaldehyde) in DPBS (Dulbecco’s modified PBS) for 15 min on RT (room temperature). Next, cells were blocked for 60 min at RT in a blocking solution (DPBS supplemented with 2 mg/mL BSA, 0.1% Triton-X 100, and 1% fish gelatin with or without 5% goat serum). Then, the samples were incubated for 60 min at RT in the blocking solution supplemented with the following primary antibodies: OCT3-4 (1:50, Santa Cruz, CA, USA, #SC-5279) and NANOG (1:100, R&D Systems, Minneapolis, MN, USA, #AF1997) as pluripotency markers; AFP (1:500, Sigma, St. Louis, MO, USA#A8452), SMA (1:500, Abcam, Cambridge, UK, #ab7817), and ß-III-tubulin (1:2000, R&D Systems, Minneapolis, MN, USA, #RD-MAB1195) as markers specific for the three lineages. After washing with DPBS, the cells were incubated for 60 min at RT with Alexa Fluor 647-conjugated goat anti-mouse and Alexa Fluor 594-conjugated donkey anti-goat IgG antibodies (1:250, ThermoFisher Scientific, Waltham, MA, USA, #A11029, #A11012). DAPI (ThermoFisher Scientific, Waltham, MA, USA, # D1306) was used for nuclear staining. The samples were then examined by a Zeiss LSM 710 confocal laser scanning microscope.