Gingiva tissue sections were imaged using super resolution confocal scanning microscopy (Zeiss 880 LSM NLO Airyscan; 1024 × 1024 pixels; magnification: 63x unless specified otherwise) and/or fluorescence microscopy (Zeiss Axio Imager A1 with QImaging MicroPublisher 3.3). The fluorescence was measured at an excitation of 488 nm and emission of 526 for green, an excitation of 561 nm and emission of 650 for red, and excitation of 405 nm and emission of 453 for blue (DAPI). For all samples, 20-35 slices of z-stacks were obtained with each slice at 0.4-0.6µm. A mid-slice within each tissue was chosen for analysis and image presentation. To process data sets, Zeiss Zen imaging software and QCapture Pro software were used for confocal/epifluorescence microscopy, respectively. Representative images were selected to visually display any differences in the antibody staining.
880 lsm nlo airyscan
Manufactured by Zeiss
The 880 LSM NLO Airyscan is a high-performance laser scanning microscope designed for advanced imaging applications. It combines the Airyscan detector technology with nonlinear optical (NLO) capabilities, enabling enhanced resolution, sensitivity, and imaging depth. The system is versatile and suitable for a wide range of research applications, but a detailed description of its intended use is not available.
Automatically generated - may contain errors
Lab products found in correlation
Axio imager a1, by Zeiss (2 mentions)
Qimaging micropublisher 3, by Zeiss (2 mentions)
Qcapture pro software, by Zeiss (2 mentions)
Zen imaging software, by Zeiss (2 mentions)
Chamber slide, by Thermo Fisher Scientific (1 mentions)
Anti flag m2, by Cell Signaling Technology (1 mentions)
Duolink in situ detection reagent, by Merck Group (1 mentions)
4 protocols using 880 lsm nlo airyscan
1
Super-Resolution Imaging of Gingiva
2
Super-Resolution Imaging of Gingival Tissue
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Visualizing FORCP Protein Localization
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pEGFP-FORCP constructs were generated by cloning the ORF of FORCP-WT or mutant ATG of ORF into pEGFP-N1 vector with Age1/Not1 sites. For pLVX-FORCP 3xFLAG constructs, full length of FORCP-WT or FORCP-mut containing 3xFLAG in its C-terminus was subcloned into lentivirus vector pLVX-PURO with EcoR1/Xba1 sites. The BRI3BP-FLAG-Myc construct was purchased from OriGene technology, USA.
For transfection and immunostaining, 293 T cells were seeded at 300,000 cells per well in a six-well plate. After 24 hr, the cells were transfected using Lipofectamine 2000 (life technology Invitrogen) according the manufacturer’s instruction. Forty-eight hours after transfection, cells were reseeded onto chamber slides (Thermo Fisher Scientific, USA) and fixed with 4% paraformaldehyde for 10 min at room temperature (RT). Fixed cells were permeabilized by 0.5% Triton X-100 for 10 min at RT and stained with primary antibodies anti-FLAG M2 (Cell Signaling, rabbit), PDI (Sigma, mouse) or anti-FORCP custom antibody (Abgent) overnight at 4°C. After washing the cells three times with PBS, secondary antibody was added and incubated at RT for 1 hr. DNA was stained with DAPI (blue). Images were taken using a confocal microscope (Zeiss LSM 880 NLO Airyscan).
For transfection and immunostaining, 293 T cells were seeded at 300,000 cells per well in a six-well plate. After 24 hr, the cells were transfected using Lipofectamine 2000 (life technology Invitrogen) according the manufacturer’s instruction. Forty-eight hours after transfection, cells were reseeded onto chamber slides (Thermo Fisher Scientific, USA) and fixed with 4% paraformaldehyde for 10 min at room temperature (RT). Fixed cells were permeabilized by 0.5% Triton X-100 for 10 min at RT and stained with primary antibodies anti-FLAG M2 (Cell Signaling, rabbit), PDI (Sigma, mouse) or anti-FORCP custom antibody (Abgent) overnight at 4°C. After washing the cells three times with PBS, secondary antibody was added and incubated at RT for 1 hr. DNA was stained with DAPI (blue). Images were taken using a confocal microscope (Zeiss LSM 880 NLO Airyscan).
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Detecting Protein Interactions by Duolink PLA
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Duolink in situ PLA enables detection, visualization, and quantification of protein interactions (< 0 nm) as an individual dot by microscopy. Interaction between HA-NFAT1 and calcineurin A was detected in Jurkat cells by PLA using Duolink in Situ detection reagents (Sigma) according to the manufacturer’s protocol with minor modifications. In brief, cells were stimulated with anti-CD3/CD28 or PMA/ionomycin on ibidi 1 μ-slides, fixed with 4% paraformaldehyde, stained with Alexa 488-conjugated wheat germ agglutinin (WGA), permeabilized with methanol, and stained with mouse anti-HA and rabbit anti-calcineurin A primary antibodies. Cells were treated with anti-mouse MINUS and anti-rabbit PLUS probes, ligated, and amplified, and detection was performed by confocal microscopy (Zeiss LSM 880 NLO Airyscan). Slides were analyzed at 80× magnification.
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