Cells were seeded onto either 96-well glass-bottom plates and grown overnight. Cells treated with zeocin for specified time and concentration were fixed with 4% formaldehyde. Then, 0.1% Triton-X was used to permeabilize the cells, followed by blocking with 3% bovine serum albumin. Cells were then incubated with the following antibodies: phospho DNA-PKcs S2056 (ab18192, Abcam, 1:200), RAD51 (ab1837, Abcam, 1:200), RPA70 (ab176467, Abcam, 1:200), cyclin E (4132S, Cell Signalling), or HA-tag (#3724, Cell Signalling, 1:100). The cells were washed and stained with the appropriate secondary antibodies: Alexa Fluor 594 (red) goat anti-rabbit (A11012, Thermo Scientific), Alexa Fluor 488 (green) goat anti-mouse (A11001, Thermo Scientific). After washing, the cells were stained with 10 μM DAPI in PBS and visualized with the Zeiss LSM 770 microscope. Images were analyzed using the ImageJ techniques previously described [72 (link)]. Cyclin E intensity was measured for each cell. Average cyclin E intensity of cells grown in media without growth factor for 4 hours was used to define the threshold of cyclin E positive. Colocalized foci appear yellow when green and red channels are merged in ImageJ.
Lsm 770 microscope
Manufactured by Zeiss
Sourced in Germany
The LSM 770 is a high-performance laser scanning microscope designed for advanced imaging applications. It features a versatile optical system, enabling the capture of high-resolution images and data. The microscope is capable of providing detailed information about the structure and function of a wide range of samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab1837, by Abcam (3 mentions)
Alexa fluor 594 red goat anti rabbit, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 488 green goat anti mouse, by Thermo Fisher Scientific (3 mentions)
Ab18192, by Abcam (2 mentions)
Ab176467, by Abcam (2 mentions)
Axio imager 2 microscope, by Zeiss (2 mentions)
Poly d lysine, by Thermo Fisher Scientific (1 mentions)
Neural tissue dissociation kit p, by Miltenyi Biotec (1 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
Nick translation kit, by Abbott (1 mentions)
Ph2ax s139, by Cell Signaling Technology (1 mentions)
Cyclin e, by Cell Signaling Technology (1 mentions)
Cyclin a, by Abcam (1 mentions)
Xrcc4, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 594 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
96 well glass bottom plate, by Cellvis (1 mentions)
Phospho dna pkcs s2056, by Abcam (1 mentions)
Alexa fluor 488 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
Ha tag, by Cell Signaling Technology (1 mentions)
6 protocols using lsm 770 microscope
1
Immunofluorescence Analysis of DNA Damage Response
2
Immunofluorescence Analysis of Rett-iPSC Organoids
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Immunofluorescence analysis was performed as previously described (Fukuda et al., 2021 (link); Motosugi et al., 2021 (link)). Briefly, the cells were fixed with 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for 20 min. The samples were permeabilized using 0.1% Triton-X for 20 min. After washing with PBS, the samples were blocked with 1.5% bovine serum albumin (BSA) in PBS for 1 h and then subjected to the first antibody reaction. After washing with PBS, the samples were subjected to the second antibody reaction and used for microscopy analysis. Images were captured using an LSM770 microscope (Carl Zeiss, Oberkochen, Germany) or an Axio Imager 2 microscope (Carl Zeiss).
For the analysis of organoids developed from Rett-iPSCs, the organoids were dissociated using Neural Tissue Dissociation Kit (P) (Miltenyi Biotec) in accordance with manufacturer’s instruction and plated on a coverslip coated with poly-d-lysine (Thermo Fisher Scientific). After 24 h, the cells were fixed and subjected to immunofluorescence analysis, as described above. Detailed information on the antibodies used in this study is provided inTable S2 .
For the analysis of organoids developed from Rett-iPSCs, the organoids were dissociated using Neural Tissue Dissociation Kit (P) (Miltenyi Biotec) in accordance with manufacturer’s instruction and plated on a coverslip coated with poly-d-lysine (Thermo Fisher Scientific). After 24 h, the cells were fixed and subjected to immunofluorescence analysis, as described above. Detailed information on the antibodies used in this study is provided in
3
RNA-FISH Visualization of XIST, XACT, and HUWE1
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Based on previous reports, RNA-FISH was performed as described previously (Fukuda et al., 2021 (link); Motosugi et al., 2021 (link)). Briefly, the cells were cultured on a coverslip and fixed by treating with 4% PFA in PBS for 20 min. The samples were permeabilized using 0.1% Triton-X for 20 min and then subjected to RNA-FISH. The probes were prepared using a nick translation kit (Abbott Laboratories, Chicago, IL, USA), with G1A (#24690; Addgene, Cambridge, MA, USA) for XIST, and the BAC clones CTD 3063K22 and PR11-975N19 for XACT and HUWE1, respectively. VECTASHIELD with DAPI was used for nuclear staining (Vector Laboratories Inc., Burlingame, CA, USA). Images were acquired using an LSM770 microscope (Carl Zeiss) or an Axio Imager 2 microscope (Carl Zeiss).
4
Quantifying Cyclin E Levels in Stressed Cells
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Cells were seeded onto either 96-well glass-bottom plates and grown overnight. Cells treated with zeocin (10 μg/mL, 10 min) were fixed with 4% paraformaldehyde. Then, 0.1% Triton-X was used to permeabilize the cells, followed by blocking with 3% bovine serum albumin. Cells were then incubated with the following antibodies: RAD51 (ab1837, Abcam, 1:200), cyclin E (4132S, Cell Signaling), pH2AX S139 (9718S, Cell Signaling), and cyclin A (ab39, Abcam). The cells were washed and stained with the appropriate secondary antibodies: Alexa Fluor 594 (red) goat anti-rabbit (A11012, Thermo Fisher Scientific), Alexa Fluor 488 (green) goat anti-mouse (A11001, Thermo Fisher Scientific). After washing, the cells were stained with 10 µM DAPI in PBS and visualized with the Zeiss LSM 770 microscope. Images were analyzed using the ImageJ techniques previously described (Murthy et al., 2018 (link)). cyclin E intensity was measured for each cell. Average cyclin E intensity of cells grown in media without growth factor for 4 hr was used to define the threshold of cyclin E positive.
5
Zeocin-Induced DNA Damage Response
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Cells were seeded onto either 96-well glass-bottom plates (Cellvis) or coverslips, and grown overnight. Cells treated with Zeocin for specified time and concentration were fixed with 4% formaldehyde. Then, 0.1% Triton-X solution in PBS was used to permeabilize the cells, followed by blocking with 3% bovine serum albumin in PBS for 30 minutes. Cells were then incubated with the following antibodies: phospho DNA-PKcs S2056 (abcam), XRCC4 (Santa Cruz Biotechnology). The cells were washed and stained with the appropriate secondary antibodies: Alexa Fluor 594 goat anti-rabbit (Thermo Scientific A11012), Alexa Fluor 488 goat anti-mouse (Thermo Scientific A11001). After washing, the cells were stained with 30 µM DAPI in PBS, and visualized with the Zeiss LSM 770 microscope. Images were analyzed using the ImageJ techniques previously described [3 (link)].
6
Immunofluorescence Imaging of DNA Repair Proteins
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Cells were seeded onto either 96-well glass-bottom plates and grown overnight. Cells treated with zeocin for specified time and concentration were fixed with 4% formaldehyde. Then, 0.1% Triton-X was used to permeabilize the cells, followed by blocking with 3% bovine serum albumin. Cells were then incubated with the following antibodies: phospho DNA-PKcs S2056 (ab18192, Abcam, 1:200), RAD51 (ab1837, Abcam, 1:200), RPA70 (ab176467, Abcam, 1:200), or HA-tag (#3724, Cell Signalling, 1:100). The cells were washed and stained with the appropriate secondary antibodies: Alexa Fluor 594 (red) goat anti-rabbit (A11012, Thermo Scientific), Alexa Fluor 488 (green) goat antimouse (A11001, Thermo Scientific). After washing, the cells were stained with 10 µM DAPI in PBS and visualized with the Zeiss LSM 770 microscope. Images were analysed using the ImageJ techniques previously described [64] (link). Colocalized foci appear yellow when green and red channels are merged in ImageJ.
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