Protoplasts were prepared from Arabidopsis rosette leaves grown under short day conditions and transformed using 30–50 μg of plasmid DNA via the PEG method (Wu et al., 2009 (link); Yoo et al., 2007 (link)). Transformed protoplasts were incubated in darkness for 36–48 hours with control (+sucrose; .5% sucrose added), starvation (–sucrose; without sucrose) or mannitol (350 mM mannitol) treatments and concanamycin A (Sigma) or dimethyl sulfoxide (DMSO) were added 12 hours before visualization by confocal microscopy. Confocal microscopy was performed with a Leica (Leica Microsystems) SP5 X MP confocal microscope equipped with a resonance scanner and HPX PL APO CS 63.0×1.40 oil objective. For colocalization assays, YFP and CFP were imaged sequentially to avoid cross-detection between channels. Excitation and detection wavelengths were the same as those described for BiFC colocalization assays.
Sp5 x mp confocal microscope
Manufactured by Leica
The Leica SP5 X MP confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a built-in multiphoton excitation capability, enabling high-resolution, deep-tissue imaging. The microscope provides researchers with a versatile and powerful tool for a wide range of biological and materials science investigations.
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6 protocols using sp5 x mp confocal microscope
1
Arabidopsis Protoplast Transformation and Microscopy
2
Live-cell imaging of BES1-GFP under starvation
3
Intracellular Trafficking of F-AuNSs
4
Retinal Immunostaining After Cryopreservation
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Dissected retinas were equilibrated at 4°C in sucrose solutions of increasing concentrations in a step-wise manner, 10%, 20%, 30% sucrose (w/v) in PBS, for at least 30 minutes or until the retina had settled to the bottom of the tube. While the retinas were in 30% sucrose/PBS, they were snap-frozen on dry ice and subjected to three freeze/thaw cycles. Retinas were stored at −80°C in the 30% sucrose/PBS solution or used for immunostaining immediately. Following freeze/thaw, retinas were rinsed 3 times in PBS for 30 minutes and blocked for 2 hours at room temperature (RT) in blocking solution [3% goat serum/1% bovine serum albumin (BSA)/0.1% Triton-X100/0.02% sodium dodecyl sulfate (SDS) in PBS]. Retinas were then incubated in primary antibody in blocking solution overnight at 4°C on a rocking platform. The next day, retinas were rinsed 3 times in PBS for 30 minutes and placed in secondary antibody in blocking solution overnight at 4°C on a rocking platform. Retinas were rinsed 3 times in PBS for 30 minutes and then flattened between two coverslips for confocal imaging on a Leica SP5 X MP confocal microscope.
5
Intracellular Trafficking of F-AuNSs
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Cells (5000 cells per well) were seeded in 8-well chamber slides (Lab-Tek) and incubated overnight. Cells were then exposed to F-AuNSs (100 μg per mL) at different time points (2, 4, 6, 8, 12, 18, and 24 h), fixed with 4% formaldehyde solution for 10 min, permeabilized with 0.1% Triton X-100 for 1 h, and blocked with 10% normal goat serum solution at room temperature for 1 h. Cells were incubated overnight at 4 °C with anti-EEA1 antibody (Abcam, ab109110, 1/250) for early endosome staining or anti-RAB7 antibody (Abcam, ab126712, 1/50) for late endosome staining, and washed three times followed by staining with Alexa Fluor 594-conjugated anti-rabbit IgG (Abcam, ab150080, 1/500). For lysosome imaging, cells were stained with BioTracker NIR 633 (Sigma, 1/500) according to the manufacturer’s instructions. Slides were mounted with DAPI nuclear dye and visualized under a Leica SP5 X MP confocal microscope. Images were captured utilizing a 63× objective.
6
Mitochondrial Membrane Potential Evaluation
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Mitochondrial membrane potential (ΔΨm) of the oocytes was evaluated using the lipophilic cationic dye JC‐1 (T3168, Thermo Fisher Scientific), as previously established (J. Dai et al., 2015 (link)). Briefly, 20 MII‐stage oocytes (three independent replicates) were incubated in 2 µM JC‐1 in TCM‐199, for 30 min at 38.5°C in 5% CO2. They were then washed with PBS to eliminate nonspecific surface fluorescence and mounted on glass slides for evaluation. Samples were visualized using a Leica SP5X MP confocal microscope using the rhodamine isothiocyanate (RITC, red) channel to identify J‐aggregates and fluorescein isothiocyanate (FITC, green) channel to identify the monomeric form of JC‐1, inside the inner mitochondrial membrane. Images of double fluorescence were recorded in oocytes with the largest diameter and analyzed using Image J software (Version 1.50; National Institutes of Health) to quantify the signal intensity of red and green fluorescence. The ratio of RITC (J‐aggregate) to FITC (J‐monomer) for each oocyte was determined and reported as the ΔΨm.
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