Live imaging was performed using a spinning disk confocal microscope (Nikon Ti2000, Yokugawa CSU-X1) equipped with 488- and 561-nm diode lasers, an EMCCD camera (Hamamatsu), and a 60× water-immersion objective (CFI Plan Apo VC 60X WI, NA 1.2, Nikon). Acquisition parameters were controlled by a home-developed LabVIEW program (LabVIEW, National Instruments). For C. elegans mitotic and female meiotic spindles, images were acquired every 2 or 4 s with a single z-plane. For human mitotic spindles, images were acquired every 2 or 4 s with three z-sections every 1 µm, and the middle planes were presented.
Ti2000
Manufactured by Nikon
Sourced in Japan
The Ti2000 is a microscope platform designed for advanced imaging and analysis in life science research. It features a modular design that supports a range of imaging techniques, including widefield, confocal, and total internal reflection fluorescence (TIRF) microscopy. The Ti2000 is capable of high-resolution image capture and supports a variety of sample types and experimental configurations.
Automatically generated - may contain errors
Lab products found in correlation
Em ccd camera, by Hamamatsu Photonics (2 mentions)
Csu x1, by Hamamatsu Photonics (2 mentions)
Cfi plan apo vc 60x wi, by Nikon (2 mentions)
Labview, by National Instruments (2 mentions)
Glass bottomed dish, by NEST Biotechnology (1 mentions)
Alexa fluor 647 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Tcs sp5 2, by Leica (1 mentions)
Imagej, by Adobe (1 mentions)
5 protocols using ti2000
1
Live Imaging of Mitotic Spindles
2
Visualizing Mitochondrial Protein Localization
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Confocal microscopy was used to investigate whether LC3, PINK1, or Parkin colocalized with the mitochondria. TOMM20, a mitochondrial outer membranous protein, was used to label mitochondria. HK-2 cells were seeded onto a glass-bottomed dish (NEST, Beijing, China) for confocal microscopic observation. After I/R treatment, HK-2 cells were rinsed twice with PBS and then fixed with 4% paraformaldehyde for 20 min at 37°C. All cells were permeabilized with 0.3% Trion X-100 for 20 min and washed with PBS. Next, the cells were blocked with 5% bovine serum albumin (BSA) and incubated overnight at 4°C in primary antibody (concentration of antibodies according to manufacturer’s instruction). The cells were rinsed twice with PBS and then further incubated with fluorescein isothiocyanate (FITC)- or tetramethylrhodamine isothiocyanate (TRITC)-conjugated secondary antibody at room temperature for 1 h. At last, 4,6-diamidino-2-phenylindole was used to dye the nuclei. The images were detected and analyzed via laser scanning confocal microscopy (Nikon, Ti2000, Japan).
3
Indirect Immunofluorescence Imaging Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were fixed (4% paraformaldehyde, 30 min at RT), permeabilized (0.1% Triton X-100, 5 min at RT), blocked (2% goat serum, 60 min at room temperature) and processed for indirect immunofluorescence. Following overnight primary antibody incubation (4 °C), cells were incubated (60 min, RT) with Alexa Fluor 488-, Alexa Fluor 568-, or Alexa Fluor 647-conjugated secondary antibodies (Invitrogen) and mounted using Mowiol. Images were captured on a confocal laser microscope system (Nikon A1R) connected to an inverse microscope (Ti-2000; Nikon) or on a Leica TCS SP5 II (Leica Microsystems) connected to an upright microscope, using an oil-immersion plan Apo 60 × A/1.40 NA objective lens. Data collected using Nikon imaging software or LAS (Leica Microsystems) were further processed with ImageJ and PhotoshopCS6 (Adobe, CA). Co-localization analysis using Pearson’s coefficient was calculated on images obtained under identical microscopy settings using ImageJ with JACoP plugin89 (link) and the data were represented as Mean ± SEM of at least 30 cells from two independent experiments.
4
Live Cell Imaging with Spinning Disk Confocal
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Live imaging was performed using a spinning disk confocal microscope (Nikon Ti2000, Yokugawa CSU-X1), equipped with 488 nm and 561 nm diode lasers, an EMCCD camera (Hamamatsu), and a 60× water-immersion objective (CFI Plan Apo VC 60X WI, NA 1.2, Nikon). Acquisition parameters were controlled using a home-developed LabVIEW program (LabVIEW, National Instruments). Images were acquired every 1 s with a single z-plane.
5
Subcellular Localization of GFP and avrBs2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For subcellular localization, the coding sequence of GFP was amplified from pGWB5 (Nakagawa et al. 2007 ) and was ligated into the pUC19 plasmid after digestion with SphI and HindIII. The 35S promoter fragment was cleaved from pUC19-35S-FLAG-RBS by EcoRI and SacI and was then religated into pUC19-GFP. The ORF of avrBs2 was amplified using the primer set avrBs2-BamHI-F and avrBs2-SalI-R and was then cloned into pUC19-35S-GFP after digestion with BamHI and SalI. The construct was confirmed by sequencing.
The transfection assay in protoplasts was carried out as described previously (Li et al. 2005) . Briefly, protoplasts were isolated from 10-day-old etiolated seedlings of Oryza sativa cv. Nipponbare or the leaves of 4-week-old NHO1-LUC transgenic Arabidopsis plants. The protoplasts (approximately 2.5 × 10 6 protoplasts per milliliter) were transfected with 10 µg of plasmid DNA by polyethylene glycol-mediated transformation. After washing with W5 solution (154 mM NaCl, 125 mM CaCl 2 , 5 mM KCl, and 2 mM MES-KOH, pH 5.7), the protoplasts were resuspended in W5 solution and were treated with 1 µM flg22 for 12 h, under low light. The LUC activity was then evaluated by measuring luminescence intensity 10 min after adding 50 µM luciferin to the transfected Arabidopsis protoplasts. GFP fluorescence on the transfected rice protoplasts was observed using confocal microscopy (Nikon Ti2000).
The transfection assay in protoplasts was carried out as described previously (Li et al. 2005) . Briefly, protoplasts were isolated from 10-day-old etiolated seedlings of Oryza sativa cv. Nipponbare or the leaves of 4-week-old NHO1-LUC transgenic Arabidopsis plants. The protoplasts (approximately 2.5 × 10 6 protoplasts per milliliter) were transfected with 10 µg of plasmid DNA by polyethylene glycol-mediated transformation. After washing with W5 solution (154 mM NaCl, 125 mM CaCl 2 , 5 mM KCl, and 2 mM MES-KOH, pH 5.7), the protoplasts were resuspended in W5 solution and were treated with 1 µM flg22 for 12 h, under low light. The LUC activity was then evaluated by measuring luminescence intensity 10 min after adding 50 µM luciferin to the transfected Arabidopsis protoplasts. GFP fluorescence on the transfected rice protoplasts was observed using confocal microscopy (Nikon Ti2000).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!