Lsm780 confocal microscope system

Immunostaining was used to look at the expression of 3-NT, matrix metalloproteinase-9, tight junction protein Claudin-5, and extracellular matrix Collagen IV in brain tissues 24 h after a stroke. Frozen brain sections were blocked with 5% goat serum (Thermo Fisher Scientific) for 1 h before being incubated with primary antibodies overnight at 4 °C: anti-3-NT (Abcam, 1:50), MMP-9 (Santa Cruz, 1:100), Claudin-5 (Thermo Scientific, 1:200), and Collagen IV (Thermo Fisher Scientific) (Abcam, 1:800). Secondary antibodies were incubated at room temperature for 2 h, including Alexa Fluor 568 Goat anti-mouse (Invitrogen), Alexa Flor 488 Goat anti-rabbit (Invitrogen), Alexa Flor 647 Goat anti-mouse (Invitrogen). The nucleus was stained with DAPI. Fluorescence signals were detected and captured using a Carl Zeiss LSM 780 confocal microscope system.

Purified exosomes were labeled with PKH26 Red Fluorescent Cell Linker Midi Kit for General Cell Membrane Labeling (#MIDI26, Sigma-Aldrich) according to the manufacturer's manual. The PKH126-labeled exosomes and the control (dyes in PBS) were washed with a large volume of PBS for three times to remove excess dyes. Labeled exosomes and control were then added to HUVECs for 12 hours. After washing twice with PBS, the cells were fixed in 4% paraformaldehyde and stained with 4',6-diamidino-2-phenylindole, dilactate (DAPI, # D-3571, Thermo Fisher Scientific) to visualize the nuclei. The fluorescent signals were examined using a Carl Zeiss LSM 780 confocal microscope system (Zeiss, Jena, Germany).

The live-cell imaging assay was performed as described previously (Giraldo et al., 2013 (link)). For vegetative hyphal imaging, a small plug of mycelium was cut from an agar culture, placed on the edge of a sterile water agar-coated microscope slide and incubated in a humid chamber for 16–18 h. For imaging invasive hyphae, leaf sheath inoculation was performed using the susceptible rice line YT16. Differential interference contrast microscopy was performed with a Zeiss Axioplan 2 IE MOT microscope, using × 40/0.75 and × 63/1.2 NA (numerical aperture) C-Apochromat water immersion objective lens. Images were acquired using a Zeiss AxioCam HRc camera and analyzed with Zeiss Axiovision digital image-processing software (version 4.8). Confocal imaging was performed with a Zeiss LSM780 confocal microscope system using two water immersion objectives, C-Apochromat 40x/1.2 W Corr and C-Apochromat 40x/1.2 W Corr. Images were acquired and processed using ZEN 2010 software.

The FRAP experiments were performed using TIE2-GFP HUVECs on glass bottomed cell culture dishes in the presence or absence of human or mouse recombinant purified angiopoietins (500 ng/ml) in environmentally controlled microscopy stage incubator. Regions of interest (ROIs) were defined and photobleached at retracting cell edges at the cell–ECM interface using the maximum laser power (405 nm) with 16 iterations. Fluorescence recovery was followed every 5 s for a period of 5 to 10 min. The following equation was used to determine the normalized fluorescence intensity (NFI): $NFI=\frac{\mathrm{R}\mathrm{O}\mathrm{I}\left(\mathrm{t}\right)-BG\left(t\right)}{\mathrm{T}\mathrm{o}\mathrm{t}\left(\mathrm{t}\right)-BG\left(t\right)}x\frac{\mathrm{T}\mathrm{o}\mathrm{t}\left({\mathrm{t}}_0\right)-BG\left(t_0\right)}{\mathrm{R}\mathrm{O}\mathrm{I}\left({\mathrm{t}}_0\right)-BG\left(t_0\right)}$ where BG stands for background, Tot for non-bleached control ROI, and t for time. The plateau point was determined as a postbleach point, after which the fluorescence signal stayed constant. The mobile fraction (Mf) was determined by: $f=\frac{{\mathrm{N}\mathrm{F}\mathrm{I}}_0+\mathrm{N}\mathrm{F}\mathrm{I}}{{\mathrm{N}\mathrm{F}\mathrm{I}}_0}$ where NFI₀ stands for the NFI in the prebleach period. Images were captured by a Zeiss LSM780 confocal microscope system with a Plan-Apochromat 40x/1.4 objective, and the time-lapse data was analyzed by ZEN 2012 (blue edition) software (Carl Zeiss).

The isolated pancreatic acinar cells, ileal crypts, or cultured CMT93 cells, treated with or without 0.5 μg/ml recombinant mouse galectin-9 for 48 h, were labeled with AO in the growth medium (1:10,000 dilution from the 2% AO stock). The cells were incubate at 37 °C for 20 min and then washed twice with live imaging solution (Invitrogen #A14291DJ). Live imaging solution was added to cover the cells in a confocal dish and immediately proceeded for microscopic analysis. Groups of cells in the brightfield (eight pre-defined areas of each well) were identified in the brightfield and lysosomes (visible as black dots) were put in focus very rapidly to avoid any damage to lysosomes since the brightfield also contains blue light. The cells were immediately and continuously exposed to 489-nm light from a 100 mW diode laser while laser scanning micrographs where captured every 484 ms on a Zeiss LSM780 confocal microscope system in two channels defined by secondary bandpass filters for 495–555 nm (green) and LP650 nm (Red) light for 180 s. Loss of lysosome integrity in cells was visualized by the loss of red fluorescence, which is normalized to unexposed cells.

Images were captured on a Zeiss LSM780 confocal microscope system using × 40 and × 63 objectives. Image manipulation was performed with ZEISS ZEN microscope software and subsequently with ImageJ.