The CaWAKL20 coding sequence (CDS) without a stop codon was amplified from the pepper line R9 using the GFP-CaWAKL20-F and GFP-CaWAKL20-R primer pair (Supplementary Table S1 ); the PCR product was then cloned into a GFP-tagged pBI221 transient expression vector. The empty vector was used as the control. Particle bombardment was used to transfect plasmid into onion epidermal cells, which were then incubated for 24 h at 28°C in the dark. The GFP signal was visualized using A1R confocal laser scanning microscopy (Nikon, Tokyo, Japan). For plasmolysis analysis, the transformed onion epidermal cells were treated in 0.8 M mannitol solution for 15 min before observation of GFP expression (Genovesi et al., 2008 (link)).
A1r laser scanning confocal microscopy
Manufactured by Nikon
Sourced in Japan
The A1R laser-scanning confocal microscopy is a high-resolution imaging system designed for scientific research. It uses a focused laser beam to scan the sample and collect optical sections, enabling the visualization of complex biological structures with improved clarity and contrast compared to traditional widefield microscopy.
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Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Facsjazz, by BD (1 mentions)
Z0458, by Agilent Technologies (1 mentions)
S3023, by Agilent Technologies (1 mentions)
Sucrose solution, by Merck Group (1 mentions)
D sorbitol, by Merck Group (1 mentions)
Alexa fluor 594 conjugated secondary antibody, by Abcam (1 mentions)
Glycerol, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Urea, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
E cadherin, by Abcam (1 mentions)
Fvmpe rs microscopy, by Olympus (1 mentions)
Perfect focus system, by Nikon (1 mentions)
11 protocols using a1r laser scanning confocal microscopy
1
Transient Expression of CaWAKL20 in Onion Cells
2
Cellular Uptake and Subcellular Localization of PVHRs Nanoparticles
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4T1 cells were seeded into 6-well plates at 1 × 105 cells/well. After culturing for 24 h, the cells were treated with C6-loaded PV micelles, PVs micelles, PVHPs NP, or PVHRs NP for 2 h39 (link). For competition assay, excess HA or RGD was added 30 min prior to C6@PVHRs NP. Cellular uptake was measured using fluorescence microscopy. To further quantify uptake efficiency, the cells were dissociated following the incubation procedure, then washed twice with PBS at 1200×g. The cells were resuspended in PBS at 1 × 106 cells/mL and analyzed using flow cytometry (FACSJazz, BD Biosciences, USA).
To evaluate the subcellular localization of PVHRs nanoparticles, 4T1 cells were seeded into 6-well plates at 1 × 105 cells/well. After culturing for 24 h, the cells were treated with PVHRs NP containing 1 μg of YOYO-1-labeled pDNA for 2 h. The cells were stained with LysoTracker Red for 30 min, then washed, fixed, and stained with Hoechst 33258 solution. The cells were visualized using A1R confocal laser scanning microscopy (Nikon, Tokyo, Japan).
To evaluate the subcellular localization of PVHRs nanoparticles, 4T1 cells were seeded into 6-well plates at 1 × 105 cells/well. After culturing for 24 h, the cells were treated with PVHRs NP containing 1 μg of YOYO-1-labeled pDNA for 2 h. The cells were stained with LysoTracker Red for 30 min, then washed, fixed, and stained with Hoechst 33258 solution. The cells were visualized using A1R confocal laser scanning microscopy (Nikon, Tokyo, Japan).
3
Immunofluorescence analysis of cellular proteins
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Transfected cells grown on glass coverslips were fixed in methanol for 5 min, followed by acetone for 20 s (−20 °C), washed in PBS, and incubated with p62CT antibody (GP62C, Progen, 1:100), K8 and 18 antibodies (#6038 and #61028, Progen, 1:100), and ubiquitin (Z0458, DAKO, 1:100) for 1 h. Coverslips were rinsed with PBS and incubated with a secondary anti-guinea pig antibody (rhodamine red) (#93431, Jackson Immune Research Laboratories, West Grove, PA, USA, 1:200) and anti-mouse Alexa fluor 488 (ThermoFisher scientific, Austria, 1:200) for 30 min at room temperature. Coverslips were washed with PBS and briefly rinsed with 70% ethanol, then air-dried and mounted with mounting medium (#S3023, DAKO). Nikon A1R confocal laser scanning microscopy was used to analyze the images.
4
Quantifying GFP Expression in Yeast
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Yeast cells containing the GFP gene were cultured in YPD medium at 30 °C for 1d and then collected by centrifugation at 5,000 rpm for 2 min and washed with phosphate buffer (PBS, 100 mM, pH 7.4). To test the relative fluorescence intensity, the samples were diluted with PBS to OD600 = 10 and were measured by a SpectraMax M5 microplate reader (excitation 488 nm; emission 520 nm) at sensitivity = 100. To observe the fluorescence changes visually, 10 μL of cell preparations were directly plated on slides and observed at 488 nm and 561 nm with a Nikon A1R confocal laser scanning microscopy (Nikon, Japan).
5
IVA Distribution in Differentiated Calu-3 Cells
6
Immunostaining and Optical Clearing of Organoids
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After being cultured for 7 days, organoids were fixed with 4.0% PFA and washed for three times using PBS (1 × ). Then, the organoids were permeabilized with 0. 5% Triton X-100 for 5 min at room temperature. After incubation in 5% bovine serum albumin (Sigma-Aldrich) for 1 h, the organoids were incubated with antibodies against E-cadherin, EpCAM or Vimentin (1:200, Abcam) at 4°C for 48 h. After washing with PBS (1 × ), the organoids were further incubated with Alexa Fluor 594 conjugated secondary antibody (1:500, Abcam). Finally, the nuclei were stained with DAPI (1:1000, Beyotime). Then, the organoids were dehydrated in 30% sucrose solution (wt/vol, sigma) at 4°C for 24h, and cleared by using the ultrafast optical clearing regents, composed of 20% urea (wt/vol, sigma), 30% D-sorbitol (wt/vol, sigma), and 5% glycerol (wt/vol, sigma) dissolved in DMSO (sigma), for 5min at room temperature before imaging. The images were captured by a Nikon A1R+ Laser scanning confocal microscopy.
7
Immunofluorescence Imaging of Cultured Cells
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Cells were cultured on glass coverslips or rat tail Collagen-I coated coverslips (Corning, cat# 354236, 40 ng/cm2) for 24–48 h. Cultured cells were fixed with 4% PFA, penetrated with 0.25% Triton X-100, blocked with 1% BSA and then incubated with antibodies. Antibodies used in this study are listed in Supplementary Table 3 . Live imaging and Z-stack imaging were conducted by Nikon A1R laser-scanning confocal microscopy with 40x objective set at 5 s time interval and 0.5 μm z-stack interval.
8
Immunostaining of Cultured Hippocampal Neurons
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Hippocampal neurons were cultured at a density of 2 × 104/cm2 in 8 mm slide for 10 days. Transfected neurons were firstly washed twice with phosphate buffer solution (PBS, 0.1 M, for all experiments, unless stated) and fixed in 4% paraformaldehyde (PFA) for 20 mins. The neurons were treated with 0.3% Triton X-100 for 10 mins at room temperature (RT), followed by sequential overnight incubations with indicated antibody at 4 °C and fluorescent secondary antibodies diluted in PBS containing 3% bovine serum albumin and 0.3% Triton X-100 for 2 hrs at RT. The fluorescent signals were examined using an A1R laser-scanning confocal microscopy (Nikon, Japan).
9
Multimodal Imaging of Cell Morphology
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Cultured cells or rehydrated paraffin bone sections were fixed with 4% PFA, penetrated with 0.25% Triton X-100, blocked with 1% BSA and then incubated with antibodies. Antibodies used in this study were listed in Table 2 . Live imaging and Z-stack imaging were conducted by Nikon A1R laser-scanning confocal microscopy with 5 sec time interval and 0.5 μm z-stack interval. The cellular morphology of MLO-Y4 cells was observed on scanning electron microscope (SEM, ZEISS Merlin) at 5.0 kV. In brief, cells growing on glass coverslips were fixed with 4% PFA and underwent sequential dehydration by incubation in a series of methanol solutions for 10 minutes per solution: 35%, 50%, 75%, 90% and 100% methanol. Samples were completely dehydrated with a series of hexamethyldisilazane (HMDS) solutions in 100% methanol for 10 minutes: 25%, 50%, 75%, and 100%. Completely dehydrated samples were left to air dry in hood overnight. Cells on coverslips were mounted with double-sided conductive tapes, and coated with Au/Pt (Gold/Platinum) particles before SEM scanning.
10
Immunofluorescence Staining of Paraffin-Embedded Bone Samples
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Immunofluorescence staining with paraffin samples were conducted as described in Refs. [39 ,40 (link)]. Specifically, paraffin bone samples were cut in 5 μm thickness, followed by standard re-hydration process, penetrated with 0.25% Triton X-100, blocked with 1% BSA and then incubated with antibodies. Antibodies used in this study are listed in Table 2 . Confocal images were conducted by Nikon A1R laser-scanning confocal microscopy with 0.5 μm z-stack interval. Second-harmonic generation (SHG) and two-photon excited fluorescence imaging were conducted to investigate collagen fibers with inverted two-photon Olympus FVMPE-RS microscopy.
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