Cells were fixed for 10 min with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS for 20 min and blocked with donkey serum (PBS with 0.3% triton, donkey serum 1:100). Cells were then stained overnight at 4 °C with anti-ɣH2AX antibody (Cell Signaling, Danvers, MA, USA; #9718, 1:400), followed by incubation for 1 h at room temperature with Alexa Flour 488-conjugated secondary antibody (Jackson Immunoresearch, Cambridge, UK; #711–545-152, 1:500) and 0.2 μg/ml 4′,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, Rehovot, Israel). Images were collected using LSM 700 laser scanning confocal system (Zeiss, Gottingen, Germany). Mean number of foci per nucleus was determined using the FIJI software with the BioVoxxel plugin.
Lsm 700 laser scanning confocal system
Manufactured by Zeiss
Sourced in Germany
The LSM 700 is a laser scanning confocal system designed for high-resolution imaging. It uses a laser as the illumination source and a pinhole aperture to achieve optical sectioning, enabling the capture of clear, in-focus images from within a specimen. The system is capable of generating detailed, three-dimensional reconstructions of samples.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Alexa flour 488 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Secondary cy 3 conjugated antibody, by Jackson ImmunoResearch (1 mentions)
Anti rad51 antibody, by Abcam (1 mentions)
Anti γh2ax antibody, by Abcam (1 mentions)
Lc3b primary antibody, by Novus Biologicals (1 mentions)
Alexa fluor 488 conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rhodamine conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Normal goat serum (ngs), by Vector Laboratories (1 mentions)
Prolong gold reagent, by Thermo Fisher Scientific (1 mentions)
Zen software, by Zeiss (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
5 protocols using lsm 700 laser scanning confocal system
1
Immunofluorescence Quantification of γH2AX
2
Quantifying DNA Double-Strand Breaks
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To assess DNA DSBs, cells grown on glass cover slips were irradiated with 4 Gy and treated with TTFields applied for 1 h, 2 h, 4 h, or 24 h. At these time points cells were fixed in freshly prepared 4% paraformaldehyde solution for 10 min. at room temperature. For staining, the fixed cells were permeabilized with 0.1% Triton X-100, and incubated with anti-γH2AX antibody (1:500) (Abcam Cambridge, GBR) or anti-Rad51 antibody (1:500) (Abcam Cambridge, GBR) at room temperature followed by incubation with secondary Cy-3 conjugated antibody (Jackson Immunoresearch West Grove, PA, USA). Slides were mounted in prolonged anti-fade solution supplemented with DAPI (Sigma Aldrich St. Louis, MO, USA). Images were collected using a LSM 700 laser scanning confocal system (Zeiss Göttingen, DEU), attached to an upright motorized microscope with a 63X/1.40 oil objective (ZeissAxio Imager Z2 Göttingen, DEU). Image analysis was carried out with Image J software (NIH, Maryland, USA).
3
Quantitative LC3 Immunofluorescence Assay
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Cells were fixed with ice-cold methanol for 10 min, serum-blocked and stained with anti-microtubule-associated protein 1 LC3B primary antibody (rabbit polyclonal, Novus Biologicals, Littleton, CO, Canada; NB600-1384, 1:200) followed by Alexa Fluor 488-conjugated secondary antibody (Jackson ImmunoResearch, Cambridge, UK; 711-545-152, 1:300) and DAPI (Sigma-Aldrich, Rehovot, Israel; 32670, 1:1000) for nuclei counterstaining. Images were collected using an LSM 700 laser scanning confocal system (Zeiss, Gottingen, Germany), attached to an upright motorized microscope (ZeissAxio Imager Z2, Gottingen, Germany). Mean number of LC3 foci per cell was determined using ImageJ software with a median filter to find the local maxima, and expressed as a percentage relative to control.
4
Immunofluorescence Staining of Cells
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Immunostaining was performed as described previously (Sasaki et al., 2015) . Briefly, cells were fixed in 1% paraformaldehyde, permeabilized with 0.5% Triton X-100 in PBS, blocked with 10% normal goat serum (Vector Laboratories) and stained with antibodies overnight at 4 C. Appropriate secondary antibodies conjugated with Alex Fluor 488, 555, or 647 (Thermo Fisher Scientific) were used for sample visualization. 4',6'-diamidino-2-phenylindole (DAPI) and rhodamine-conjugated phalloidin (Thermo Fisher Scientific) were used to visualize nuclei and filamentous actin, respectively. Samples were mounted with Prolong Gold reagent (Thermo Fisher Scientific) and examined at room temperature using an AxioImager Z1 microscope equipped with an LSM700 laser scanning confocal system (Carl Zeiss) and a water-immersed objective lens (C-Apochromat 40 3 /1.2W, Carl Zeiss). Images were processed and analyzed with the Zen software (Carl Zeiss) and were assembled using Photoshop software (Adobe Systems).
5
Imaging of E. coli BL21(DE3) Protein Complexes
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E. coli BL21(DE3) cells co-expressing Ct-MOMP and Bam were fixed with 0.8% formaldehyde at 4 °C overnight. The cells were gently washed 3x with ice-cold PBST (0.05% Tween 20) and harvested by centrifugation (5000× g, 5 min, 4 °C). To permeabilize membranes and peptidoglycan, the cells were resuspended in PBS, pH 7.4, containing 0.1% Triton X-100, 100 µg/mL lysozyme, 5 mM EDTA and incubated at room temperature for 45 min [66 (link)]
After blocking with PBS containing 3% BSA for 1 h, cells were incubated with primary antisera (anti-MOMP, 1:1000 or anti-BamB, 1:200) for 1 h, subsequently with secondary antiserum (Alexa488 goat anti-rabbit, 1:200) 1 h at RT. Then, cells were immobilized by a thin layered 1% agarose on glass slides. The phase contrast and fluorescence images of cells were obtained by using LSM700 laser scanning confocal system from Zeiss (Florida, USA) and further processed using ImageJ software (http://rsb.info.nih.gov/ij/ ).
After blocking with PBS containing 3% BSA for 1 h, cells were incubated with primary antisera (anti-MOMP, 1:1000 or anti-BamB, 1:200) for 1 h, subsequently with secondary antiserum (Alexa488 goat anti-rabbit, 1:200) 1 h at RT. Then, cells were immobilized by a thin layered 1% agarose on glass slides. The phase contrast and fluorescence images of cells were obtained by using LSM700 laser scanning confocal system from Zeiss (Florida, USA) and further processed using ImageJ software (
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