U2OS cells were transfected with NT, FANCD2 or FANCG siRNA. Cells were incubated overnight and then treated with the appropriate damaging agent. Cells were xed, blocked, and permeabilized. Cells were stained using the following antibodies: anti-BRCA1 (sc-6954) from Santa Cruz Biotechnologies, anti-53BP1 (#4937) from Cell Signalling, anti-phospho DNA-PKcs (ab18192) from AbCam, or anti-Mre11 (GTX118741) from GeneTex. Secondary antibodies AlexaFluor 596-labeled donkey anti-rabbit IgG, AlexaFluor 488-labeled chicken anti-mouse IgG (Molecular Probes) were used. Images were obtained using a Zeiss LSM 510 Meta scanning confocal microscope and were analyzed using ImageJ software. At least 250 nuclei were counted per experiment.
Lsm510 meta scanning confocal microscope
Manufactured by Zeiss
Sourced in Germany
The LSM510-Meta scanning confocal microscope is a high-performance imaging system designed for advanced scientific research. It features a modular architecture that allows for customization and integration of various imaging modalities. The core function of the LSM510-Meta is to provide high-resolution, three-dimensional imaging of samples through optical sectioning and confocal detection.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield mounting medium, by Vector Laboratories (2 mentions)
Anti brca1 sc 6954, by Santa Cruz Biotechnology (1 mentions)
Aim software version 4, by Zeiss (1 mentions)
Anti vimentin antibody, by Agilent Technologies (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Cx41 microscope, by Canon (1 mentions)
Powershot s5 is camera, by Canon (1 mentions)
5 protocols using lsm510 meta scanning confocal microscope
1
Immunofluorescence Assay for DNA Damage Markers
2
Immunofluorescence Assay for Nuclear Stress Bodies
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To detect nuclear stress bodies, immunofluorescence was performed as in Sandqvist et al. (2009 (link)). Briefly, HeLa cells were cultured on coverslips and fixed with 100% methanol for 6 min at 4 °C. The methanol was aspirated, and the cells were washed three times with 0.05% PBS-Tween20. The cells were incubated in a blocking solution containing 10% BSA (bovine serum albumin) in 0.05% PBS-Tween20 for 1 h. The cells were incubated with anti-HSF1 antibody (Holmberg et al. 2000) diluted in blocking solution (1:300) overnight at 4 °C. The secondary antibody (anti-rabbit IgG, Alexa Fluor 488) was diluted in blocking solution (1:700) and added for 1 h. The coverslips were mounted using VECTASHIELD mounting medium (Vector Laboratories), and the cells were visualized with an LSM510-Meta scanning confocal microscope (Carl Zeiss).
3
Immunofluorescence Staining of Vimentin
4
Visualizing Mouse Lung Histology and Bacterial Internalization
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Mouse lungs were fixed overnight in 4% paraformaldehyde before being embedded in paraffin. Slides of paraffin sections were stained with hematoxylin and eosin (H&E) and imaged on an Olympus Cx41 microscope and images captured using a Canon Powershot S5IS camera.
BMDC were incubated with exponential phase bacteria labeled with 1 mM of pHrodo (Life Technologies) for 2 h. Fluorescein isothiocyanate phalloidin (Life Technologies) and TO-PRO3 (Life Technologies) were incubated in the presence of 0.2% Triton X-100 prior to fixing in 4% paraformaldehyde and visualization with a Zeiss LSM 510 META scanning confocal microscope
BMDC were incubated with exponential phase bacteria labeled with 1 mM of pHrodo (Life Technologies) for 2 h. Fluorescein isothiocyanate phalloidin (Life Technologies) and TO-PRO3 (Life Technologies) were incubated in the presence of 0.2% Triton X-100 prior to fixing in 4% paraformaldehyde and visualization with a Zeiss LSM 510 META scanning confocal microscope
5
Detecting Nuclear Stress Bodies via Immunofluorescence
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To detect nuclear stress bodies, immunofluorescence was performed as in (Sandqvist et al. 2009) (link).
Briefly, HeLa cells were cultured on coverslips and fixed with 100% methanol for 6 min at 4 °C. The methanol was aspirated and the cells were washed three times with 0.05% PBS-Tween20. The cells were incubated in a blocking solution containing 10% BSA (bovine serum albumin) in 0.05% PBS-Tween20 for 1 h. The cells were incubated with anti-HSF1 antibody (Holmberg et al. 2000) diluted in blocking solution (1:300) overnight at 4 °C. The secondary antibody (anti-rabbit IgG, Alexa Fluor 488) was diluted in blocking solution (1:700) and added for 1 h. The coverslips were mounted using VECTASHIELD mounting medium (Vector Laboratories) and the cells were visualized with an LSM510-Meta scanning confocal microscope (Carl Zeiss).
Briefly, HeLa cells were cultured on coverslips and fixed with 100% methanol for 6 min at 4 °C. The methanol was aspirated and the cells were washed three times with 0.05% PBS-Tween20. The cells were incubated in a blocking solution containing 10% BSA (bovine serum albumin) in 0.05% PBS-Tween20 for 1 h. The cells were incubated with anti-HSF1 antibody (Holmberg et al. 2000) diluted in blocking solution (1:300) overnight at 4 °C. The secondary antibody (anti-rabbit IgG, Alexa Fluor 488) was diluted in blocking solution (1:700) and added for 1 h. The coverslips were mounted using VECTASHIELD mounting medium (Vector Laboratories) and the cells were visualized with an LSM510-Meta scanning confocal microscope (Carl Zeiss).
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