Tissue samples collected from the anterior wall of LV were preserved with 2.5% glutaraldehyde for 24 h at 4 °C and handled as previously reported [16 (link)]. Images were taken using a transmission electron microscope (TEM) (JEM-1230, JEOL Ltd., Tokyo, Japan) at 300 kV. Image-Pro Plus 6.0 software was used to determine the number and size of mitochondria. The morphometric analysis includes at least 300 mitochondria from at least eight pictures per heart. The proportion of mitochondria in a specific area categorized into three size groups (< 0.6 μm2, within 0.6–1.0 μm2, >1.0 μm2) was counted as previously described [6 ,17 (link)]. MitoTrackerRed CMXRos probe (100 nmol/L, Invitrogen, Carlsbad, USA, 30 min at 37 °C) was used to label mitochondria in the cells. Nikon A1R MP + confocal laser-scanning microscope was used to take the images. The number of mitochondria per cell and mitochondrial size were counted and measured as we previously described [10 ].
A1r mp confocal laser scanning microscope
Manufactured by Nikon
Sourced in Japan
The A1R-MP confocal laser scanning microscope is a high-performance imaging system designed for advanced biological research. It features a multi-photon excitation capability, enabling deep tissue imaging with reduced photodamage. The microscope provides high-resolution, high-speed imaging capabilities, making it a versatile tool for a wide range of applications in the life sciences.
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Lab products found in correlation
Mitotracker red cmxros probe, by Thermo Fisher Scientific (1 mentions)
Jem 1230, by JEOL (1 mentions)
Dmem f 12, by Nacalai Tesque (1 mentions)
Can get signal immunostain, by Toyobo (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
X tremegene 9 dna transfection reagent, by Roche (1 mentions)
Htert rpe1 cells, by American Type Culture Collection (1 mentions)
Nis elements ar software, by Nikon (1 mentions)
Minimal essential medium, by Nacalai Tesque (1 mentions)
Axiovert 200mat microscope, by Zeiss (1 mentions)
Fugene 6, by Promega (1 mentions)
5 protocols using a1r mp confocal laser scanning microscope
1
Quantitative Analysis of Cardiac Mitochondria
2
Quantitative Analysis of hiPSC Neural Differentiation
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To measure the neural differentiation of hiPSCs, immunocytochemistry was performed. The fixed cells were incubated with primary antibodies and then with secondary antibodies. The information of antibodies is shown in Supplementary Table S5 . Immunofluorescence images were acquired using a Nikon A1R-MP confocal laser scanning microscope (Nikon). To investigate the distribution of ligand on culture substrate, vitronectin-coated area was quantified using confocal images. Detail is described in Supplementary methods.
3
Inducing and Analyzing Cilia Formation
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The hTERT-RPE1 cells (CRL-4000; American Type Culture Collection) were cultured in DMEM/F-12 (Nacalai Tesque) supplemented with 10% FBS and 0.348% sodium bicarbonate. To induce ciliogenesis, cells were grown to 100% confluence on dishes or coverslips and starved for 24 h in Opti-MEM (Invitrogen) containing 0.2% bovine serum albumin. Expression vectors were transfected into the cells using X-tremeGENE9 DNA Transfection Reagent (Roche Applied Science).
Immunofluorescence analysis was performed as described previously (Takahashi et al., 2012 (link); Nozaki et al., 2017 (link)). Cells were fixed with 3% paraformaldehyde at 37°C for 15 min, washed three times with phosphate-buffered saline (PBS), quenched with 50 mM NH4Cl for 10 min, washed three times with PBS, permeabilized with 0.1% Triton X-100 for 5 min, and washed three times with PBS. For the detection of endogenous RABL2, cells were fixed with 10% trichloroacetic acid on ice for 15 min. The fixed/permeabilized cells were blocked with 10% FBS and treated with antibodies diluted in 5% FBS. For the detection of γ-tubulin, antibodies were diluted with Can Get Signal immunostain (Toyobo). The stained cells were observed using an Axiovert 200 M microscope (Carl Zeiss) or an A1R-MP confocal laser-scanning microscope (Nikon).
Immunofluorescence analysis was performed as described previously (Takahashi et al., 2012 (link); Nozaki et al., 2017 (link)). Cells were fixed with 3% paraformaldehyde at 37°C for 15 min, washed three times with phosphate-buffered saline (PBS), quenched with 50 mM NH4Cl for 10 min, washed three times with PBS, permeabilized with 0.1% Triton X-100 for 5 min, and washed three times with PBS. For the detection of endogenous RABL2, cells were fixed with 10% trichloroacetic acid on ice for 15 min. The fixed/permeabilized cells were blocked with 10% FBS and treated with antibodies diluted in 5% FBS. For the detection of γ-tubulin, antibodies were diluted with Can Get Signal immunostain (Toyobo). The stained cells were observed using an Axiovert 200 M microscope (Carl Zeiss) or an A1R-MP confocal laser-scanning microscope (Nikon).
4
Multimodal Confocal Microscopy Imaging
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The scanning was performed using a A1R MP+ confocal laser scanning microscope (Nikon, Shinagawa, Tokyo, Japan). The 405-nm and 561-nm emission lasers and the following optics were used in the present study: Plan Apo 20x/0,75 Dic N, Apo IR 60x/1,27 WI and Apo TIRF 60x/1,49 oil Dic lens (Nikon, Shinagawa, Tokyo, Japan). The cell contours were visualized using differential interference contrast. The images obtained were processed using the NIS-Elements AR software (Nikon).
5
Imaging HeLa Cell Protein Localization
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HeLa cells were cultured in minimal essential medium (Nacalai Tesque) supplemented with 10% heat-inactivated fetal bovine serum at 37°C under a 5% CO2 atmosphere. HeLa cells were transfected with expression plasmids for CDC50A and ATP10 proteins, ATP11 proteins, or their chimeric constructs using FuGENE6 (Promega), and incubated for 48 h. HeLa cells were transfected with Lyn11-EGFP–fused constructs or HA-tagged ATP10B-NT and ATP11B-CT constructs using FuGENE6 (Promega), and incubated for 24 h. Immunostaining was performed as described previously (Shin et al., 2004 (link); Takatsu et al., 2014 (link)) and was visualized using an Axiovert 200MAT microscope (Carl Zeiss) and an A1R-MP confocal laser scanning microscope (Nikon). Briefly, HeLa cells were fixed with 3% paraformaldehyde and then permeabilized with 0.2% saponin for 20 min or 0.1% Triton X-100 for 5 min at room temperature. The cells were blocked with 10% fetal calf serum in phosphate-buffered saline (PBS) at room temperature for 30 min and incubated sequentially with primary and secondary antibodies at room temperature for 1 h. Coverslips were placed using Mowiol mounting medium.
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