Tcs sp8 confocal microscope

For the determination of cell surface localization of nucleolin in AFAP-GFP-expressing HeLa cells, HeLa cells transfected with pAFAP-GFP for 3 d were subjected to incubation at RT for 1 h with mouse monoclonal anti-nucleolin antibody (MS-3, SCBT) diluted in DMEM medium at 1:20. After washing with DMEM medium 3 times, cells were fixed with 4% paraformaldehyde at RT for 20 min. Fixed cells were washed 3 times with 1 × PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, and 2 mM KH₂PO₄, pH 7.4) and blocked in 1 × PBS containing 0.8% BSA for 10 min, followed by incubation at 37 °C for 45 min with Alexa Fluor 555-conjugated goat anti-mouse antibody (Invitrogen). These immunolabeled cells were observed under a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany). Other cells transfected with fluorescent protein plasmids were fixed with 4% paraformaldehyde at RT for 20 min and subjected to observation under a Leica TCS SP8 confocal microscope (Leica) or a Nikon Eclipse Ti2 confocal microscope.

To determine the localization of type-B OsRRs in plant cells, we transfected rice protoplasts with OsRR–GFP fusion constructs. Red fluorescent protein (RFP) with a nuclear localization sequence (NLS) was used as nucleus-localized control. GFP fluorescence (493–546 nm) was observed and imaged using a Leica TCS SP8 confocal microscope (Leica Microsystems, Germany). For BiFC assays, OsRR17 was fused to the N-terminus of the Venus variant of GFP (OsRR17-VN), while three OsHPs were fused to the C-terminus of Venus (OsHP-VC). The corresponding constructs were transfected in rice protoplasts as above. OsbZIP10-VC was used as a negative control, and NLS-RFP was used as a nucleus-localized control. RFP fluorescence (581–652 nm) was observed and imaged using a Leica TCS SP8 confocal microscope.

The localization of GFP‐ and mCherry‐labelled proteins in fresh tissue samples was captured by a Leica TCS SP8 confocal microscope (Leica, Bensheim, Germany). GFP was excited with Argon laser at 488 nm and the emitted fluorescence was detected between 493 and 550 nm. mCherry was excited at 561 nm and the emitted fluorescence was detected between 576 and 632 nm. Homozygous transgenic plants carrying the CDKF;1–GFP–PIPL, CDKD;1–GFP, CDKD;2–GFP–PIPL, CDKD;3–GFP and CYCH–GFP constructs were germinated on vertical MSAR agar plates and grown for 10 days in a controlled culture room. The roots of seedlings were stained for 30 sec with 0. 01% PI (propidium iodide, Sigma‐Aldrich) followed by washing several times with water. GFP and PI fluorescence of root tips was captured by a Leica TCS SP8 confocal microscope. PI was excited at 488 and the emitted fluorescence was detected between 600 and 657 nm. Merging of images was performed using the Leica LAS X software. Line intensity profiles of selected regions of interest (ROI) were generated using the Image J2 software (Rueden et al., 2017) and the data were exported to Excel.

For LDL uptake cell-based assay, the cells were treated with LDL-DyLight^TM 550 (Cayman chemical, Ann Arbor, Michigan, USA) working solution in serum-free medium. Incubate the cells at 37 °C with 5% CO₂ for an additional 18 hours. At the end of LDL uptake incubation, the culture medium was replaced with fresh culture medium. The degree of LDL uptake was examined under a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany) with filters capable of measuring excitation and emission wavelengths 540 and 570 nm, respectively.
For LDL receptors examination, the cells were washed with TBS briefly, and fixed with cell-based assay fixative solution (Cayman chemical) for 10 minutes. Wash the cells with TBST three times for five minutes each. Incubate the cells for one hour with rabbit anti-LDL receptor antibody (Cayman chemical). Wash the cells with TBST three times for five minutes each. Incubate the cells for one hour with Dylight^TM 488-conjugated secondry antibody (Cayman chemical). Wash the cells with TBST three times for five minutes each. The nuclei were counter-stained with DAPI (Sigma-Aldrich). The stained cells were examined under a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany).

Immunofluorescence images of the stained cells were examined and acquired under a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany). For DPP4 density measurements in the cells, 10 fluorescence images from each coverslip were randomly captured using Leica TCS SP8 confocal microscope equipped with HC PL Apo×63/1.4 oil immersion CS2 objective. Immunofluorescence density analysis was performed using Image J software (National Institutes of Health, Bethesda, MD, USA). The relative expression of protein was presented with fold over the control.

After three and seven days, cell-laden spongy-like hydrogels were incubated with calcein-AM (Ca-AM, 1 μg/mL, Invitrogen, Carcavelos, Portugal) and propidium iodide (PI, 2 μg/mL, Invitrogen, Carcavelos, Portugal) for 1 h at 37 °C in a humidified tissue culture incubator with 5% CO₂ atmosphere. Then, constructs were fixed and counterstained with DAPI (0.02 mg/mL) and the percentage of live/dead cells was assessed with a Leica TCS SP8 confocal microscope. The number of live and dead cells for each condition was quantified in five random images (three independent experiments) using the Cell Counter plugin of FIJI for ImageJ. The percentage of live cells was calculated as followed (Equation (3)):
For visualization of the cytoskeleton F-actin fibers and nuclei, cells were fixed and stained with phalloidin-TRITC (0.1 mg/mL, Sigma-Aldrich, Loures, Portugal) and DAPI (0.02 mg/mL), respectively. Samples were observed with a Leica TCS SP8 confocal microscope.