Cells were grown on coverslips placed in 6-well plate and treated with 100 μM esculetin. Treated cells were fixed with 2 % PFA (Paraformaldehyde) at room temperature for 15 mins. Fixed cells were permeablized with perm buffer (0.2 % saponin and 0.1 % BSA), blocked with 5 % BSA and incubated overnight in anti-Nrf2 antibody at 4 °C and then in fluorescein (FITC) conjugated secondary antibody for 2 h at room temperature. After washing the cells, they were fixed with 4 % PFA and stained with nuclear stain DAPI (4′,6-diamidino-2-phenylindole). Confocal imaging was performed with Nikon C2 laser scan confocal microscope with 60× objective magnification, numerical aperture 1.4, refractive index 1.5, Plan Apo optics equipped with an argon laser, using excitation and emission wavelength of 490 and 525 nm respectively for FITC and 461 and 500 nm respectively for nuclear stain DAPI (4′,6-diamidino-2-phenylindole). Data was analyzed using the NIS Elements AR software.
C2 laser scan confocal microscope
Manufactured by Nikon
Sourced in Japan
The Nikon C2 laser scan confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It utilizes a laser-based scanning mechanism to capture detailed images of microscopic samples. The C2 microscope offers high-resolution, multi-channel imaging capabilities, enabling researchers to visualize and analyze a variety of specimens with precision.
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Lab products found in correlation
6 protocols using c2 laser scan confocal microscope
1
Nrf2 Localization under Esculetin Treatment
2
Immunofluorescence Staining of L-type Ca2+ Channels
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At the end of incubation cells were fixed with acetone:methanol::1∶1 for 20 min in 4°C. Cells were washed twice and incubated with antibodies against L-type Ca2+ α1C for 2h followed by addition of anti-rabbit FITC tagged Alexa flour 488 for 1.5h. Cells were again washed and mounted with anti-fade containing DAPI. Confocal imaging was performed on Nikon C2 laser scan confocal microscope (Nikon, Japan) with 60X objective magnification, numerical aperture 1.4, refractive index 1.5, Plan Apo optics equipped with Argon laser, using excitation and emission wavelength of 488 nm, respectively. Data were analyzed using the NIS Elements AR software.
3
PspA3-286 Binding to Dendritic Cells
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DCs (2 x 106/ml) were incubated with recombinant PspA3–286 from R36A [38 (link)]. Cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% saponin, and incubated with antibodies against PD-L1 tagged with phycoerythrin. Cells were fixed with 4% paraformaldehyde. Confocal imaging was performed with Nikon C2 laser scan confocal microscope with 60x objective magnification, numerical aperture 1.4, refractive index 1.5, Plan Apo optics equipped with an argon laser, using excitation and emission wavelength of 405 and 488 nm, respectively. Data were analyzed using the NIS Elements AR software.
4
Visualizing BCG Infection in Macrophages
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This was carried out essentially as described recently (22). Briefly, 2-D08 treated or Sumo1 or Ubc9 knockdown BMDCs were infected with 2.5 MOI BCG for 24. These BMDCs were co-cultured with BCG primed T cells for 48h. In parallel, J774 mouse macrophages were infected with 10 MOI GFP-expressing BCG for 24h. Following this T cells enriched from DC:T cell co-culture were layered onto GFP-BCG infected macrophages and incubated for 72h. Confocal imaging was performed with Nikon C2 laser scan confocal microscope with 60x magnification. Data were analyzed using NIS Elements AR software.
5
Immunofluorescence Imaging of CACNA1S
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At the end of incubation cells were fixed with acetone: methanol 1:1 for 20 min in 4°C. Cells were washed twice and incubated with antibodies against CACNA1S for 2h followed by addition of anti-rabbit FITC tagged Alexa flour 488 for 1.5h. Cells were again washed and mounted with anti-fade containing DAPI. Confocal imaging was performed on Nikon C2 laser scan confocal microscope (Nikon, Japan) with 60X objective magnification, numerical aperture 1.4, refractive index 1.5, Plan Apo optics equipped with Argon laser, using excitation and emission wavelength of 488 nm, respectively. Data were analyzed using the NIS Elements AR software.
6
Monitoring Intracellular Mycobacterial Survival
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Monitoring of intracellular mycobacterial survival was carried out as recently described [21] . Briefly, BMDCs were stimulated with 10 µM Bcl2 Inhibitor. After 1h the cells were infected with 10 MOI of GFP expressing M. bovis BCG for 72h. Cells were washed with 1X PBS post 72 h of infection to remove any extracellular bacteria. Cells were mounted on slides with Fluoroshield mounting medium containing DAPI. Single plane confocal images were acquired using a Nikon C2 laser scan confocal microscope at 60X objective magnification, a numerical aperture of 1.4, and a refractive index of 1.5. Data were analyzed using the NIS Elements Advanced Research software. The MFI numbers in bar graphs of confocal microscopy images represent Mean Fluorescence Intensity (of FITC Channel) of three experiments for all the bacteria present in multiple fields per experiment as calculated by ROI Statistics option present in the toolbar of NIS Elements Analysis Software. This is calculated by the software itself.
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