Human keratinocytes were cultured to 70% confluence on glass coverslips and then fixed on ice in either methanol for 2 min or 4% paraformaldehyde for 10 min followed by 0.2% Triton X-100 for 7 min. Primary antibodies described above were detected with Alexa Fluor-conjugated secondary antibodies. Wide field fluorescence microscopy was performed using a DMRXA2 microscope (Leica, Wetzler, Germany) equipped with a 63×/1.32 NA oil immersion objective and narrow band pass filters. Images were acquired with an ORCA digital camera (Hamamatsu Photonics, Bridgewater, NJ) and processed using Simple PCI software (Hamamatsu Corporation, Sewickley, PA). Super-resolution microscopy was performed using a Nikon N-SIM system on an Eclipse Ti-E microscope system equipped with a 100×/1.49 NA oil immersion objective, 488- and 561-nm solid-state lasers in 3D structured illumination microscopy mode. Images were captured using an EM charge-coupled device camera (DU-897, Andor Technology) and reconstructed using NIS-Elements software with the N-SIM module (version 3.22, Nikon). Colocalization analysis was performed by obtaining Mander’s coefficient using ImageJ plugin JACoP [35] (link).
N sim module
Manufactured by Nikon
Sourced in United Kingdom
The N-SIM module is a component of Nikon's advanced microscopy systems. It enables super-resolution imaging, which allows for the visualization of cellular structures and processes at a resolution beyond the diffraction limit of traditional light microscopy. The core function of the N-SIM module is to provide high-resolution imaging capabilities to researchers and scientists.
Automatically generated - may contain errors
Lab products found in correlation
Du897, by Oxford Instruments (4 mentions)
Dmrxa2 microscope, by Leica (2 mentions)
Eclipse ti e microscope system, by Nikon (2 mentions)
N sim system, by Nikon (2 mentions)
Nis elements software, by Nikon (1 mentions)
Alexa fluor 488 goat anti chicken, by Thermo Fisher Scientific (1 mentions)
Prolong glass antifade mountant, by Thermo Fisher Scientific (1 mentions)
Anti vimentin, by BioLegend (1 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (1 mentions)
5 protocols using n sim module
1
Immunofluorescence Imaging of Human Keratinocytes
2
Immunofluorescence Staining and Super-Resolution Microscopy
3
Widefield and Super-Resolution Microscopy
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Widefield fluorescence microscopy was performed using a DMRXA2 microscope (Leica, Wetzler, Germany) equipped with a 100×/1.40 NA oil immersion objective and narrow band pass filters. Images were acquired with an ORCA digital camera (Hamamatsu Photonics, Bridgewater, NJ) and processed using Fiji ImageJ. Superresolution microscopy was performed using a Nikon N-SIM system on an Eclipse Ti-E microscope system equipped with a 100×/1.49 NA oil immersion objective, 488- and 561-nm solid-state lasers in three-dimensional (3D) SIM mode. Images were captured using an EM charge-coupled device camera (DU-897; Andor Technology) and reconstructed using NIS-Elements software with the N-SIM module (version 3.22; Nikon). All microscopy was performed at room temperature. Widefield microscopy results are representative of two independent replicates with at least 10 cells each, whereas SIM results are representative of at least 50 desmosomes per condition.
4
Immunofluorescence Staining and Super-Resolution Microscopy
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Patient tissue slices were allowed to come to room temperature and immunostained with primary and secondary antibodies for 1 hr each at room temperature with triple PBS+ washes between antibody incubations. HKs in Figure 1 were fixed in methanol and processed for immunofluorescence. Primary antibodies described above and patient IgG present in tissues was detected with Alexa Fluor-conjugated secondary antibodies. Widefield fluorescence microscopy was performed as previously described (Stahley et al., 2014 (link)). Super-resolution structured illumination microscopy (SIM) was performed using the N-SIM system equipped with a 100x/1.49 NA oil immersion objective and 488- and 561-nm solid-state lasers. 3D SIM images were captured with an EM charge-coupled device camera (DU-897, Andor Technology, UK) and reconstructed using NIS-Elements software with the N-SIM module (version 3.22, Nikon, Melville, NY). Colocalization analysis via Mander's coefficient was performed using ImageJ Fiji and plugin JACoP (Bolte and Cordelieres, 2006 (link)).
5
3D Structured Illumination Microscopy of Vimentin
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Mouse embryonic fibroblasts are seeded on #1.5 glass coverslips and fixed with 4% PFA for 10 min at RT. The fixed cells are permeabilized with 0.1% Triton-X 100 for 10min at RT and stained with anti-vimentin (1:200, Biolegend, CA, USA) for 30 min in PBS containing 5% normal goat serum (RT). This is followed by incubation with goat anti-chicken Alexa Fluor 488 (1:400, Invitrogen, CA) and Alexa Fluor 568 phalloidin (1:400, Invitrogen, CA) in PBS for 30 min. (RT).
The coverslips containing the stained cells are mounted with ProLong Glass antifade mountant (Life Technologies, Carlsbad, CA, USA) on microscope slides. 3D SIM is carried out with a Nikon N-SIM Structured Illumination microscope system (Nikon N-SIM, Nikon, Tokyo, Japan) using an oil immersion objective lens (CFI SR Apochromat 100x, 1.49 NA, Nikon). For 3D SIM, 10 optical sections are imaged at 100 nm intervals in the periphery of the cell. Raw SIM images are reconstructed with the N-SIM module of Nikon Elements Advanced Research with the following parameters -Illumination contrast: 1.00; high-resolution noise suppression: 0.75; out-of-focus blur suppression: 0.25. Brightness and contrast are adjusted for image presentation.
The coverslips containing the stained cells are mounted with ProLong Glass antifade mountant (Life Technologies, Carlsbad, CA, USA) on microscope slides. 3D SIM is carried out with a Nikon N-SIM Structured Illumination microscope system (Nikon N-SIM, Nikon, Tokyo, Japan) using an oil immersion objective lens (CFI SR Apochromat 100x, 1.49 NA, Nikon). For 3D SIM, 10 optical sections are imaged at 100 nm intervals in the periphery of the cell. Raw SIM images are reconstructed with the N-SIM module of Nikon Elements Advanced Research with the following parameters -Illumination contrast: 1.00; high-resolution noise suppression: 0.75; out-of-focus blur suppression: 0.25. Brightness and contrast are adjusted for image presentation.
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