DNA fiber assay was performed as described in Bellelli et al. (2018) (link). Briefly, Rtel1F/FPole4+/+and Pole4−/− MEFs infected with CRE recombinase or GFP expressing adenovirus, were pulse labeled with 20 μM CldU for 20 min and subsequently with 200 μM IdU for 20 min. Cells were trypsinized, washed in PBS and resuspended at a concentration of 5x 105 in PBS. 2.5 μL of cell suspension were spotted on clean glass slides and lysed with 7.5 μL of 0.5% SDS in 200 mM Tris-HCL, pH 7.4, 50 mM EDTA for 10 min at R.T. Slides were then tilted allowing a stream of DNA to run slowly down the slide, air-dried and then fixed in methanol/acetic acid (3:1) for 15 min at R.T. After denaturation in HCl 2,5 M (30 min R.T.) slides were blocked in 1% BSA/PBS and incubated with rat anti-BrdU monoclonal antibody (1:1000 overnight; AbD Serotec) and mouse anti-BrdU monoclonal antibody (1:500 1h R.T.; Becton Dickinson). After washes in PBS, slides were incubated with Alexa Fluor 488 rabbit anti-mouse and Alexa Fluor 594 goat anti-rat antibodies (1:500 R.T.; Invitrogen) for 45 min and mounted in PBS/Glycerol 1:1. Fibers were then examined using Axio Imager.M2 (ZEISS) with 60x oil immersion objective and the Volocity 6.3 software.
Alexa fluor 594 goat anti rat antibodies
Manufactured by Thermo Fisher Scientific
Alexa Fluor 594 goat anti-rat antibodies are fluorescent-labeled secondary antibodies used for detection and visualization in various immunoassays and imaging applications. They specifically bind to primary antibodies raised in rats, allowing for the detection and localization of target antigens.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti brdu monoclonal antibody, by BD (4 mentions)
Rat anti brdu monoclonal antibody, by Bio-Rad (3 mentions)
Alexa fluor 488 rabbit anti mouse, by Thermo Fisher Scientific (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Axio imager m2, by Thermo Fisher Scientific (1 mentions)
Rat monoclonal anti brdu antibody, by Abcam (1 mentions)
4 protocols using alexa fluor 594 goat anti rat antibodies
1
DNA Fiber Assay for Replication Dynamics
2
DNA Fiber Assay for Chromatin Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA fiber assay was performed as described in(Bellelli et al., 2018 (link)). Briefly, eHAP ALC1+/+ and ALC1−/− transfected with control siRNA or siRNA targeting BRCA1 or BRCA2, were pulse labeled with 20 μM CldU for 20 min and subsequently with 200 μM IdU for 20 min. After tripsinization and counting, cells were resuspended at a concentration of 5x 105 in PBS and 2.5 μL of cell suspension were spotted on glass slides and lysed with 7.5 μL of a buffer containing 0.5% SDS, 200 mM Tris-HCL, pH 7.4, and 50 mM EDTA. Slides were then tilted to allow a stream of DNA to move slowly toward the botton of the slide, briefly air-dried and then fixed in methanol/acetic acid (3:1) (15 min at R.T). Slides were subsequently denatured in HCl 2,5 M (30 min R.T.), extensively washed in dH2O and PBS, blocked in 1% BSA/PBS (30 min R.T.) and incubated with rat anti-BrdU monoclonal antibody (1:1000 overnight; AbD Serotec) and subsequently with mouse anti-BrdU monoclonal antibody (1:500 1 h R.T.; Becton Dickinson). After incubation with a mixture of Alexa Fluor 488 rabbit anti-mouse and Alexa Fluor 594 goat anti-rat antibodies (1:500 45 min R.T.; Invitrogen) slides were mounted in PBS/Glycerol 1:1 and finally examined using Axio Imager.M2 (ZEISS) with 63x oil immersion objective and the Volocity 6.3 software.
3
Visualizing DNA Replication Dynamics
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA fibre assays were essentially performed as described in Bellelli et al (2018) (link). Briefly, U2OS cells transfected with siRNAs against histones H1 or a control siRNA were pulse-labelled with 20 μM CldU for 20 min and subsequently with 200 μM IdU for 20 min. Cells were trypsinized, washed in PBS, and resuspended at a concentration of 5 × 105 in PBS. 2.5 μl of cell suspension was spotted on clean glass slides and lysed with 7.5 μl of 0.5% SDS in 200 mM Tris–HCl, pH 7.4, and 50 mM EDTA for 10 min at R.T. Slides were then tilted allowing a stream of DNA to run slowly down the slide, air-dried, and then fixed in methanol/acetic acid (3:1) for 15 min at R.T. After denaturation in HCl 2.5 M (30 min, R.T.), slides were blocked in 1% BSA/PBS and incubated with a rat anti-BrdU monoclonal antibody (1:1,000, overnight; Abcam) and a mouse anti-BrdU monoclonal antibody (1:500, 1 h, R.T.; Becton Dickinson). After washes in PBS, slides were incubated with Alexa Fluor 488 rabbit anti-mouse and Alexa Fluor 594 goat anti-rat antibodies (1:500, R.T.; Invitrogen) for 45 min and mounted in PBS/glycerol (1:1). Fibres were then examined using the Zeiss LSM 710 confocal microscope with 63x oil immersion objective and ImageJ software.
4
DNA Fiber Assay for Replication Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA fiber assay was essentially performed as described in Bellelli et al., 2018a (link)). Briefly, MEFs of the indicated genotypes were pulse labelled with 20 μM CldU for 20 min and subsequently pulse labelled with 200 μM IdU for 20 min. Cells were trypsinized, washed in PBS, counted and resuspended at a concentration of 5 × 105 in PBS. 2.5 μL of cell suspension were spotted on clean glass slides and lysed with 7.5 μL of 0.5% SDS in 200 mM Tris-HCL, pH 7.4, 50 mM EDTA (10 min, R.T.). Slides were tilted (15° to horizontal), allowing a stream of DNA to run slowly down the slide, air dried and then fixed in methanol/acetic acid (3:1) for 15 min at R.T. Acid-treated slides (45 min R.T.) were blocked in 1% BSA/PBS for 45 min at R.T. and incubated with rat anti-BrdU monoclonal antibody (1:1,000 over night; AbD Serotec) and mouse anti-BrdU monoclonal antibody (1:500 1h R.T.; Becton Dickinson). After 3 washes in PBS, slides were incubated with a mixture of Alexa Fluor 488 rabbit anti-mouse and Alexa Fluor 594 goat anti-rat antibodies (1:500 R.T.; Invitrogen) for 45 min at room temperature and mounted in PBS/Glycerol 1:1. Fibers were then examined using Axio Imager.M2 (ZEISS) with 60x oil immersion objective and the Volocity 6.3 software. For quantification at least 500 replication structures were counted per experiment.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!