Tcs sp2 confocal laser scanning microscope

All fluorescent images were generated using a Leica TCS SP2 Laser Scanning Confocal Microscope (Cambridge Advanced Imaging Centre, Cambridge, UK). Images were captured using a x40/1.1 or a x63/1.2 water immersion objectives. Dylight 405, Alexa Fluor 488 and TdTomato (568) were excited with 405, 488, and 561 nm laser lines and emission collected with 395 to 415 nm, 500 to 550 nm and 580 to 620 nm bandpass emission filters, respectively. All images were captured using sequential scanning mode and image stacks were collected with 1.0 μm focus intervals. Eight bit confocal images were acquired with a 512 x 512 pixels format, a scan speed of 400 Hz. All images were digitally processed with Adobe Photoshop (Adobe System), where the levels of brightness and contrast were adjusted to enhance the quality of images. As for chromagen labelled sections, images were acquired using a Zeiss Axio Imager A1 microscope (Centre for Trophoblast Research, Cambridge, UK). The number of PRV-GFP-stained or other antibody-stained neurons/cell bodies was counted in each region of two brain sections for each animal and indicated as mean number with standard error of mean (SEM).

Images of anther semi-thin sections were visualized and photographed using an Olympus BX51 microscope equipped with an Olympus DP 70 digital camera. For confocal microscopy, samples were observed with a Leica TCS SP2 laser scanning confocal microscope using a 63×/1.4 water immersion objective lens. A 488-nm laser was used to excite GFP, EYFP, chlorophyll, and FM4-64. Emission were captured using PMTs set at 505–530 nm, 500–550 nm, and 660–760, 644–719 nm, respectively. For visualization of cell membranes and trafficking vesicles, roots were treated with 20 μM FM4-64 (Invitrogen) or 30 μM BFA (Sigma-Aldrich) for 5–30 min, washed, and then observed. For localization of TPD1 and EMS1, anthers were dissected from young buds and mounted in water. For immunogold-labeling, images were obtained with an FEI CM 120 electron microscope.

Protoplasts for transfection were obtained from suspension-cultured tobacco (Nicotiana tabacum) BY-2 cells according to the method described in Wang et al. (2005 (link)). Typically 0.2 ml of protoplast suspension was transfected with 50 µg of either 35S:EgrIAA13:GFP or 35S::GFP (control) construct. Transfected protoplasts were incubated for 16 h at 25 °C and examined for GFP fluorescence signals using a Leica TCS SP2 laser scanning confocal microscope. Images were obtained with a 40 × water immersion objective. All transient expression assays were independently repeated three times.

The effect of Danu on cellular autophagy was further examined using confocal microscopy with the application of Cyto-ID^® autophagy detection kit according to the manufacture’s instruction. The C13 and A2780cp cells were seeded into 8-well chamber slide at 30% confluence. After incubation overnight, the cells were treated with Danu at 0.01, 0.1, and 0.5 μM. After incubation for 24 h, the cells reached ~60% of confluence and were washed with 1×assay buffer, following by incubation with 100 μL of microscopy dual detection reagent for 30 min at 37 °C in the dark. After incubation, the cells were washed with 1× assay buffer to remove the detection reagent, and then examined using a TCS SP2 laser scanning confocal microscope (Leica, Wetzlar, Germany) using a standard fluorescein isothiocyanate filter set for imaging the autophagic signal at wavelengths of 405/488 nm.

Cells were seeded on glass coverslips, fixed in 3% paraformaldehyde, and then treated as previously described [43 (link)]. The primary antibodies used were: mouse monoclonal anti-CD93 (mAb 4E1, 0.6 μg/μL [5 (link)]) 1:25, rabbit anti-β-DG (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-phospho-Cbl (Y774) (Millipore, Billerica, MA, USA). Fluorescent images were captured using a Leica TCS SP2 laser-scanning confocal microscope. The quantitative colocalization analyses of CD93 and β-DG or phospho-Cbl signals were performed on optical sections captured at cell/substrate adhesion sites using ImageJ and the JACoP plug-in to determine Manders' coefficient M₁ [44 (link)], which represents the percentage of CD93 pixels that overlap β-DG or phospho-Cbl pixels. To show colocalization events by white dots, images were generated using ImageJ and the Colocalization plug-in.

The subcellular localizations of LncRNA RP11-79H23.3 and miR-107 were examined by FISH kit (Roche Applied Science, Penzberg, Germany). Cells grew to 70% confluence, were fixed with 4% formaldehyde for 15 min, then permeabilized with 0.5% TritonX-100 for 15 min and rinsed with PBS. Cells were incubated in a mixture of RP11-79H23.3 probes labeled with Cy3 at 37 °C overnight and washed with prewarmed 2× saline-sodium citrate (SSC). The FITC-labeled miR-107 probes were incubated in prehybridization buffer (1:100) at 88 °C for 5 min and at 4 °C for 3 min with the PCR instrument (Bio-Rad, Hercules, CA, USA). Next, the FITC-labeled miR-107 probes were added into cells at 37 °C overnight, washed with 2× SSC, and stained by DAPI. Observations were undertaken with a Leica TCS-SP2 Laser Scanning Confocal Microscope.