IVC and IN VIVO embryos at the blastocyst stage (ten embryos per group) were fixed with 4% paraformaldehyde (PFA), permeabilized in 0.1% Triton X-100, and then stained with 5 µg/mL of Hoechst 33,342. Stained blastocysts were visualized using a confocal microscopy (Nikon Eclipse Ti-E), using NIS-Elements Confocal software (Nikon). Images were analyzed using IMARIS 6.0.1 software (Bitplane AG, UK). Nuclei were identified using the ‘spot’ option in the software, with an estimated diameter of 7–10 μm in the Hoechst channel. The number of nuclei identified by the software was adjusted manually.
Nis elements confocal software
Manufactured by Nikon
Sourced in Japan
NIS-Elements Confocal software is a comprehensive imaging and analysis platform designed for confocal microscopy. It provides tools for image acquisition, processing, and analysis, enabling researchers to capture high-quality confocal images and extract valuable data from their samples.
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Lab products found in correlation
Eclipse ti e, by Nikon (2 mentions)
Af6000 epifluorescence microscope, by Leica (1 mentions)
Ti e inverted confocal microscope, by Nikon (1 mentions)
Las x software, by Nikon (1 mentions)
Mitotracker green fm, by Thermo Fisher Scientific (1 mentions)
A1rsi, by Nikon (1 mentions)
Cfi plan apo lambda 20, by Nikon (1 mentions)
Cfi plan fluor 40 oil, by Nikon (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
7 protocols using nis elements confocal software
1
Blastocyst Nuclei Quantification
2
Fluorescence Microscopy Imaging Protocol
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All fluorescence images were acquired using either a Leica AF6000 epifluorescence microscope coupled to a Leica DFC360 digital camera running Leica LAS-X software, or a Nikon Ti: E Inverted confocal microscope running Nikon NIS-Elements Confocal software.
3
Algal Lipid Body Quantification
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The number of algal cells with and without lipid bodies was counted along with the total number of lipid bodies present per ten fields of view [75 (link), 76 (link)]. An average lipid body per cell was determined for every 1024 × 1024 8-bit frame. The average size of lipid bodies per frame was also measured by Nikon confocal software per field of view to compare the treated algal cells with the control [73 (link), 77 (link)]. The Nikon NIS-Elements confocal software counted and summed all the pixels in the chosen fields that were above the selected threshold of brightness, thereby computing the total area of the above-threshold entities [72 (link)]. The threshold was adjusted by highlighting lipid bodies; if they varied in brightness, the least bright lipid body in the selected field served as the threshold baseline [72 (link)]. The output of each calculation yielded the average lipid body area per frame. It should be noted that measurement of lipid body area underestimates the spherical volume and hence will also underestimate the yield. It will be a comparison of lipid body areas that allows for accurate assessment of the relative yields from different samples [72 (link)].
4
Measuring Mitochondrial Membrane Potential
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JC9 is a cationic dye which binds to mitochondria and emits green fluorescence (∼525 nm) independent of mitochondrial membrane potential (MMP). In the case of mitochondrial membrane hyperpolarization, JC9 aggregates and gives off red fluorescence (∼590 nm). Therefore, the intensity ratio of red: green can be used to measure the MMP and indicates the mitochondrial activity25 (link).
IN VIVO and IVC embryos were loaded with M2 medium containing JC-9 dye (final concentration: 2 μg/ml) and incubated at 37 °C for 25 min. Then, embryos were washed twice with PBS, counterstained with 5 μg/mL Hoechst 33,342, and observed under confocal microscopy (Nikon Eclipse Ti-E), using NIS-Elements Confocal software (Nikon). After incubation blastocysts were live- imaged under a 63X objective lens with a zoom factor of 1.6X. Both green and red channel images were captured. ImageJ software was used to measure the intensity of green and red fluorescent signals and the ratio of red/green fluorescent intensity was calculated.
IN VIVO and IVC embryos were loaded with M2 medium containing JC-9 dye (final concentration: 2 μg/ml) and incubated at 37 °C for 25 min. Then, embryos were washed twice with PBS, counterstained with 5 μg/mL Hoechst 33,342, and observed under confocal microscopy (Nikon Eclipse Ti-E), using NIS-Elements Confocal software (Nikon). After incubation blastocysts were live- imaged under a 63X objective lens with a zoom factor of 1.6X. Both green and red channel images were captured. ImageJ software was used to measure the intensity of green and red fluorescent signals and the ratio of red/green fluorescent intensity was calculated.
5
Mitochondrial Dynamics in Early Embryo
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Mitochondrial distribution and segregation were quantified using Mitotracker Green staining. 2-cell and blastocysts were incubated with 100 µM of Mitotracker Green FM (Invitrogen, Molecular Probes, Milan, Italy) in M2 medium for 30 min at 37.5 °C. Then, embryos were washed twice with PBS, and then counterstained with 5 µg/mL of Hoechst 33342 for 10 min. Mitochondria distribution in embryo cytoplasm appeared as small individual fluorescent dots or as aggregates. All samples were examined by confocal microscopy (Nikon A1R), using NIS-Elements Confocal software (Nikon). Images were analyzed using IMARIS 6.0.1 software (Bitplane AG, UK).
6
Confocal Imaging of GCaMP6f, Reelin, and Wfs1
7
Multicolor Confocal Imaging of Immune Cells
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Images were acquired using an inverted Nikon A1+ confocal microscope with NIS-Elements confocal software (Nikon Instruments, Tokyo, Japan). Images were collected at digital scan resolution 0.62µm/pixel, pixel dwell 4.8 with 1024 resolution using a 20x Plan Apo dry objective (N.A. 0.75). Sequential laser scanning was performed using four lasers; 405nm (450/50 filter), 488nm (525/50 filter), 561nm (595/50 filter) and 640nm (700/75 filter) to view nuclei, chemokine receptors, nerve fibres and CD14 + or CD3 + cells respectively. The position of the top and bottom of the image was recorded before multiple images were taken in a z-series, collected according to Nyquist criteria. Equivalent thresholds were applied across images to visualise nerves (white), chemokine receptors (red) and CD14 + or CD3 + (green). Chemokine receptors co-located with either CD14 + or CD3 + cells appeared yellow.
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