γ-H2AX foci were detected using immunofluorescence as described previously [27 ]. Briefly, cells were seeded into each well of a 6-well plate with a glass coverslip. At harvest, cells were fixed in 4% paraformaldehyde for 10 min, washed three times in Tris-HCl (50 mM, pH 8.0), and incubated for 30 min with blocking buffer (0.02% Triton X-100, 10% horse serum, and 150 mM NaCl in Tris-HCl (50 mM, pH 8.0)). Glass coverslips were then incubated with primary rabbit anti-γ-H2AX polyclonal antibodies (ab11174, Acam, Cambridge, UK; 1:1000 dilution in blocking buffer) at room temperature for 1 h, washed three times in washing buffer (0.02% Triton X-100 and 200 mM NaCl in Tris-HCl (50 mM, pH 8.0)), and incubated with secondary Alexa Fluor 488 goat anti-rabbit IgG antibodies (ab150077, Abcam, Cambridge, UK; 1:300 dilution in blocking buffer) and nuclear counterstaining reagent Hoechst33342 (Abcam, Cambridge, UK) at room temperature for 1 h. Coverslips were mounted with Fluoroshield reagent (ab104135, Abcam, Cambridge, UK). Foci were visualized on a Leica DMI6000 microscope with 100× immersion objective. γ-H2AX foci were counted visually or using ImageJ. More than 100 cells in each experimental group were chosen for γH2AX quantitation.
Fluoroshield reagent
Manufactured by Abcam
Sourced in United Kingdom
Fluoroshield reagent is a water-based mounting medium designed for the preservation and protection of fluorescently-labeled samples. It is intended to be used in conjunction with fluorescence microscopy techniques.
Automatically generated - may contain errors
Lab products found in correlation
Dmi6000 microscope, by Leica (2 mentions)
Hoechst 33342, by Abcam (1 mentions)
Dmi6000, by Leica (1 mentions)
Ab150077, by Abcam (1 mentions)
Ab150131, by Abcam (1 mentions)
Ab8637, by Abcam (1 mentions)
Ab228551, by Abcam (1 mentions)
Ab9136, by Abcam (1 mentions)
Ab150105, by Abcam (1 mentions)
Ab150116, by Abcam (1 mentions)
3 protocols using fluoroshield reagent
1
Immunofluorescence Detection of γ-H2AX Foci
2
Immunofluorescence Staining of Cas9 and HBc
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells seeded on glass coverslips were fixed in 4% paraformaldehyde for 10 min. Next, coverslips were washed three times in 50 mM Tris-HCl (pH 8.0), incubated for 30 min with blocking buffer (0.02% Triton X-100, 10% horse serum, and 150 mM NaCl in 50 mM Tris-HCl [pH 8.0]), and incubated with primary goat polyclonal anti-6XHis-tag antibodies to detect StСas9-6XHis-tag protein (ab9136, Abcam) and with mouse monoclonal anti-HBc antibodies (ab8637, Abcam) at room temperature for 1 h. The cells were washed three times for 5 min in washing buffer (0.02% Triton X-100 and 200 mM NaCl in 50 mM Tris-HCl [pH 8.0]), then incubated with secondary Alexa Fluor 647 donkey anti-goat immunoglobulin G (IgG) H&L (ab150131, Abcam) and Alexa Fluor 488 donkey anti-mouse IgG H&L antibodies (ab150105, Abcam). Alternatively, coverslips were stained only with primary mouse monoclonal anti-HBc antibodies (ab8637, Abcam) and secondary Alexa Fluor 594 goat anti-mouse IgG H&L antibodies (ab150116, Abcam). Cell nuclei were counterstained with Hoechst 33342 (1/10,000; ab228551, Abcam) at room temperature for 1 h. Coverslips were washed three times for 5 min in washing buffer and mounted with Fluoroshield reagent (Abcam). Images were captured using a Leica DMI6000 microscope with 100× immersion objectives. The research was done using equipment of the Core Centrum of Institute of Developmental Biology RAS.
3
DNA Damage Immunofluorescence Imaging
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were seeded on glass coverslips, treated appropriately, and fixed in 4% paraformaldehyde for 10 min on the day of harvest, as previously described. Next, coverslips were washed 3 times in Tris-HCl (50 mM, pH 8.0), incubated for 30 min with blocking buffer (0.02% of Triton X-100, 10% horse serum, and 150 mM NaCl in Tris-HCl, 50 mM, pH 8.0), and incubated with primary rabbit anti-yH2AX antibodies (ab11175) at room temperature for 1 h. The cells were washed 3 times for 5 min in washing buffer (0.02% of Triton X-100 and 200 mM NaCl in Tris-HCl, 50 mM, pH 8.0), then incubated with secondary Alexa Fluor 488 goat anti-rabbit IgG antibodies (ab205718) with Hoechst33342 (1/10,000; ab228551) at room temperature for 1 h. Coverslips were washed 3 times for 5 min in washing buffer and mounted with Fluoroshield reagent (Abcam). Images were captured using a Leica DMI6000 microscope with 20× and 100× immersion objectives.
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!