Primary tail fibroblasts were seeded onto an 8-well chamber slide (Nunc Lab-Tek II Chamber Slide system) for overnight culture at 37 °C and infected with a m.o.i. = 5 of MCMV-M45mutRHIM mutant virus for the indicated times. ThT (25 μM) was added to cells 1 h before fixation18 (link). Cells were fixed with 4% paraformaldehyde for 15 min, permeabilized in 0.1% Triton X-100 in PBS for 10 min and blocked with 0.5% BSA in PBS containing 0.1% saponin at room temperature. Cells were then incubated with the indicated antibodies overnight at 4 °C, followed by washing with PBS-T three times at room temperature and incubation with anti-mouse IgG Cascade Blue, anti-mouse IgG Alexa Fluor 568, anti-rabbit IgG Alexa Fluor 488 or anti-rabbit IgG Alexa Fluor 568 (Thermo Fisher) at 1:1,000 dilution. The coverslips were mounted and counterstained using ProLong Gold Antifade mountant with 4,6-diamidino-2-phenylindole (DAPI, Thermo Fisher). All images were captured using identical settings on a Nikon laser scanning confocal microscope or a Leica SP8 confocal microscope with a ×60 oil-objective, and processed using Nikon’s NIS elements, Leica’s LAS X and ImageJ software.
Sp8 confocal microscope
Manufactured by Nikon
The Nikon SP8 confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a confocal scanning system that enables optical sectioning and high-resolution imaging of samples. The SP8 provides users with precise control over various imaging parameters, such as laser intensity, detection wavelengths, and scanning speed, allowing for the capture of detailed, high-quality images.
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Lab products found in correlation
Confocal laser scanning microscope, by Leica camera (1 mentions)
Lab tek 2 chamber slide system, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti rabbit igg alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade mountant with 4 6 diamidino 2 phenylindole dapi, by Thermo Fisher Scientific (1 mentions)
Las x, by Leica camera (1 mentions)
Nis element, by Nikon (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Ab13970, by Abcam (1 mentions)
A1 confocal microscope, by Nikon (1 mentions)
Tie pfs a1r confocal microscope, by Nikon (1 mentions)
4 protocols using sp8 confocal microscope
1
MCMV-M45mutRHIM Infection Assay with ThT Staining
2
Confocal Imaging of Hippocampal Sections
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Confocal microscopy image stacks were captured using a Leica SP8 confocal microscope and Nikon A1R. For images covering the entire hippocampal section, a 10× lens was used to acquire Z-stacks with a 5 µm interval at a resolution of 750 nm per pixel. For high-magnification images, a 63× lens was used to acquire Z-stacks with a 1 µm interval at a resolution of 120 nm per pixel. Multichannel fluorescence images were saved individually for analysis and merged together for co-localization studies using the Leica LAS AF or Nikon NIS Elements software suite.
3
Cardiac cell size analysis in mice
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Mouse heart samples were fixed in 4% paraformaldehyde for 3.5 hours, rinsed in PBS for 30 min, and immersed in PBS containing 30% sucrose overnight at 4°C. Heart samples were embedded in OCT for cryosectioning. Cryosections were stained with a chicken anti-GFP primary antibody (Abcam, ab13970) and a goat anti-chicken Alexa Fluor 488 (Life Technologies, A-11039) as previously described [1 (link)], followed by imaging. For whole-mount imaging, the left ventricles of uninjured hearts and infarcts were carefully removed and trimmed before imaging. Imaging was performed using a Leica SP8 confocal microscope or a Nikon A1 confocal microscope. Images obtained from cryosections were used for cell size measurement using ImageJ. Only EGFP+ cells with Dapi signals were included in cell size measurement. In case that cell boundaries between adjacent EGFP+ cells could not be identified, the entire EGFP+ area divided by the number of EGFP+ cells in this area was used as the average EGFP+ cell size in this area. Three samples per group were included in the analysis. At least three random images were used to calculate the average EGFP+ cell size of each sample.
4
Confocal Microscopy Imaging Protocol
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