HeLa cells, cultured on coverslips, underwent a 15-min pulse-labeling with 10 μM BrdU (Sigma-Aldrich) before exposure to 4 mM HU for 3 h. After washing with PBS, cells were permeabilized with 0.5% Triton X-100 for 5 min at 4 °C and fixed with 3% paraformaldehyde for 10 min at room temperature. After three PBS washes, the fixed cells were incubated sequentially with anti-BrdU antibody for 30 min and rhodamine-conjugated goat anti-mouse IgG for 30 min. DAPI was used to counterstain the nuclear DNA, and images were captured with a fluorescence Microscope (Eclipse 80i; Nikon) equipped with a Plan Fluor 60 × oil objective lens (NA 0.5–1.25; Nikon) and a camera (CoolSNAP HQ2; Photometrics).
Plan fluor 60 oil objective lens
Manufactured by Nikon
The Plan Fluor 60× oil objective lens is a high-magnification objective designed for use in microscopy applications. It features a numerical aperture of 1.40 and a working distance of 0.21 mm, providing excellent optical performance and resolution. The lens is optimized for fluorescence imaging and can be used with oil immersion techniques to enhance image quality.
Automatically generated - may contain errors
Lab products found in correlation
Eclipse 80i, by Nikon (5 mentions)
Nis elements basic research imaging software, by Nikon (3 mentions)
Bromodeoxyuridine (brdu), by Merck Group (2 mentions)
Fluorescein isothiocyanate or rhodamine conjugated second primary antibodies, by Jackson ImmunoResearch (2 mentions)
Duolink in situ red starter kit, by Merck Group (2 mentions)
Eclipse 80i fluorescence microscope, by Nikon (2 mentions)
Mouse anti brdu antibody, by GE Healthcare (1 mentions)
9 protocols using plan fluor 60 oil objective lens
1
BrdU Pulse-Labeling and Immunofluorescence
2
Immunofluorescence Staining of Cells
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Cells cultured on coverslips were fixed by 4% paraformaldehyde for 10 minutes at room temperature and then extracted with 0.5% Triton X-100 solution for 5 minutes. After blocking with Tris-buffered saline and Tween 20 solution containing 1% bovine serum albumin, cells were incubated with indicated primary antibodies for 1 hour at room temperature. After that, cells were washed and incubated with fluorescein isothiocyanate or rhodamine-conjugated second primary antibodies (1:3000 dilution, Jackson ImmunoResearch) for 1 hour. Cells were counterstained with 100 ng/mL 4′,6-diamidino-2-phenylindole (DAPI) for 2 minutes to visualise nuclear DNA. The cover slips were mounted onto glass slides with anti-fade solution and visualised on a Nikon ECLIPSE E800 fluorescence microscope with a Nikon Plan Fluor 60× oil objective lens (NA 1.30).
3
Immunofluorescence Microscopy of IR and CPT-Treated HeLa Cells
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HeLa cells cultured on coverslips were treated with 10 Gy IR or 0.5 μM CPT for the indicated times. Cells were then washed with PBS, pre-extracted with 0.5% Triton X-100 solution for 5 min, and fixed with 3% paraformaldehyde for 10 min at room temperature. After blocking with 1% BSA, cells were incubated with indicated primary antibodies for 20 min at room temperature. Subsequently, cells were washed three times with PBS and incubated with indicated secondary antibodies for 20 min at room temperature. After that, cells were stained with DAPI and visualized by a fluorescence microscope (Eclipse 80i; Nikon) equipped with a Plan Fluor ×60 oil objective lens (NA 0.5–1.25; Nikon) and a camera (CoolSNAP HQ2 (link); PHOTOMETRICS).
4
Proximity Ligation Assay for EdU-labeled HeLa Cells
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HeLa cells were seeded onto coverslips in a 6-well plate and allowed to incubate for 24 h. Following this, cells were labeled with 10 μM EdU for 15 min followed by exposure to 4 mM HU for 3 h. After PBS washing, the cells were permeabilized with 0.5% Triton X-100 at 4 °C for 10 min, fixed with a solution of 3% formaldehyde and 2% sucrose for 10 min, and blocked with 3% BSA for 30 min. After three PBS washes, the cells were subjected to a “Click-iT reaction”. Following that, cells were incubated with primary antibodies at 4 °C overnight. The proximity ligation assay was performed using the Duolink In Situ Red Starter kit (Sigma-Aldrich) following the manufacturer’s instructions. Images were captured using a fluorescence Microscope (Eclipse 80i; Nikon) equipped with a Plan Fluor 60 × oil objective lens (NA 0.5–1.25; Nikon) and a camera (CoolSNAP HQ2; Photometrics).
5
Quantifying DNA Replication Stress Response
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U2OS cells were pulse-labeled with 10 μM EdU for 15 min. Subsequently, the cells were either left untreated or treated with 4 mM HU for 3 h before they were washed with PBS. Next, the cells were permeabilized with 0.5% Triton X-100 for 5 min, fixed with a solution of 3% formaldehyde and 2% sucrose in PBS at room temperature for 10 min, and blocked with 3% BSA in PBS for 30 min. After washing with PBS, the cells were subjected to Click-iT reaction to attach biotin to EdU, and then incubated overnight at 4°C with the appropriate primary antibodies. The proximity ligation assay was performed using the Duolink In Situ Red Starter kit (Sigma-Aldrich) according to the manufacturer's instructions. Images were acquired using a Nikon Eclipse 80i Fluorescence Microscope equipped with a Plan Fluor 60× oil objective lens (NA 0.5–1.25; Nikon) and a camera (CoolSNAP HQ2; Photometrics), and analyzed with NIS-Elements basic research imaging software (Nikon).
6
Detecting Nascent ssDNA in HeLa Cells
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To detect nascent ssDNA, HeLa cells were subjected to a pulse-labeling with 10 μM BrdU (Sigma-Aldrich) for 20 min prior to treatment with 2 mM HU and 2 μM VE-821 for 3 h. Following a PBS wash, cells were permeabilized with 0.5% Triton X-100 for 5 min at 4 °C and subsequently fixed with 3% paraformaldehyde for 10 min at room temperature, without undergoing any DNA denaturation treatment. Fixed cells were then incubated with mouse anti-BrdU antibody (GE, RPN202, 1:1000) for 20 min at room temperature, followed by Rhodamine-conjugated goat anti-mouse IgG. To visualize nuclear DNA, counterstaining was performed with DAPI. Image capture was conducted using a Nikon Eclipse 80i Fluorescence Microscope equipped with a Plan Fluor 60× oil objective lens (NA 0.5–1.25; Nikon) and a camera (CoolSNAP HQ2; Photometrics), and subsequent analysis was carried out utilizing NIS-Elements basic research imaging software (Nikon).
7
Immunofluorescence Staining of Cells
8
Immunofluorescence Staining for PCNA and SIVA1
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Indirect immunofluorescence was performed as previously described (Huang et al., 2009 (link); Liu et al., 2010 (link); Wan et al., 2013 (link)). HeLa or XP30RO (gift from C. Guo, Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, China) cells cultured on coverslips were treated with 50 J/m2 UV and recovered for the indicated times. Cells were then washed with PBS, preextracted with buffer containing 0.5% Triton X-100 for 5 min, and fixed with 3% paraformaldehyde for 10 min at room temperature. For PCNA and SIVA1 coimmunofluorescence staining, HeLa cells were denatured by 2.5 M HCl for 1 h at room temperature after 3% paraformaldehyde fixation. Cells were then incubated in primary antibody for 30 min at room temperature. After three 5-min washes with PBS, secondary antibody was added at room temperature for 30 min. Cells were then stained with DAPI to visualize nuclear DNA. Images were captured with use of a fluorescence microscope (Eclipse 80i; Nikon) equipped with a Plan Fluor 60× oil objective lens (NA 0.5–1.25; Nikon) and a camera (CoolSNAP HQ2; Photometrics). Images were captured using NIS-Elements basic research imaging software (Nikon) and analyzed using Photoshop CS3 (Adobe).
9
Immunofluorescence Imaging of DNA Damage
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Cells were cultured on coverslips and were treated with 1 μM CPT or 10 Gy X-rays for the indicated times. Cells were then washed with PBS, permeabilized with PBS buffer containing 0.5% Triton X-100 for 5 min at room temperature, and were subsequently fixed with 3% paraformaldehyde for 10 min at room temperature. Coverslips were then blocked with 5% milk for 10 min before incubation with primary antibodies for 20 min at room temperature. Coverslips were washed three times with PBS before they were incubated with secondary antibodies for an additional 20 min at room temperature. Coverslips were then incubated with 4′,6-diamidino-2-phenylindole (DAPI) to visualize nuclear DNA. Images were captured with use of a fluorescence microscope (Eclipse 80i; Nikon) equipped with a Plan Fluor 60× oil objective lens (NA 0.5–1.25; Nikon) and a camera (CoolSNAP HQ2; PHOTOMETRICS).
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