48 hours after chronic treatment with 5 μM cisplatin with and without 2 μM JH-RE-06, wild-type and SMARCAL1KO U2OS cells were fixed with 4% PFA for 10 min, washed three times in PBS, permeabilized in 0.5% Triton X-100 for 10 min at RT, and blocked in BSA-T (5% BSA, 0.05% Tween 20) for at least 1 hour at RT. Immunostaining of the 53BP1 nuclear bodies was performed with rabbit anti-53BP1 antibody (1:1,000; Novus Biologicals, NM100-304S) and mouse anti-cyclin A antibody (1:100; Santa Cruz, SC-271682) in a humid chamber at 4°C overnight. Slides were then washed three times in PBS. Secondary antibodies anti-mouse Alexa Fluor 594 (1:1,000; Thermo Fisher Scientific, A11005) and anti-rabbit Alexa Fluor 488 (1:1,000; Thermo Fisher Scientific, A11034) were incubated with cells for 1 hour at RT in the dark. Slides were again washed three times in PBS. Nuclei were stained with 0.05 μg/mL DAPI for 10 min at RT. The slides were mounted with Prolong Gold Antifade reagent (Invitrogen, P36930). Images were captured with 40x objective, and at least 150 cyclin A-negative cells were analyzed per condition (Wood et al., 2020 ).
Rabbit anti 53bp1 antibody
Manufactured by Novus Biologicals
Sourced in United States
Rabbit anti-53BP1 antibody is a primary antibody that recognizes the 53BP1 protein. 53BP1 is a DNA damage response protein that plays a role in DNA double-strand break repair. This antibody can be used for applications such as Western blotting, immunoprecipitation, and immunofluorescence.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Nm100 304s, by Santa Cruz Biotechnology (1 mentions)
Mouse anti cyclin a antibody, by Santa Cruz Biotechnology (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Anti rabbit alexa 488 secondary antibody, by Thermo Fisher Scientific (1 mentions)
Axio imager z2 microscope, by Zeiss (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Sp5 confocal microscope, by Leica (1 mentions)
3 protocols using rabbit anti 53bp1 antibody
1
Quantifying DNA Damage Response in Cisplatin-Treated Cells
2
Immunofluorescence Analysis of DNA Damage Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tumor masses were fixed in 10% neutral buffered formalin at RT for 24 h and immersed in 30% sucrose/PBS at 4 °C twice, until they were sunk. They were embedded in OCT and stored at − 80 °C. Sections were cut at 10 μm and cells were permeabilized with 0.5% Triton X-100 and blocked in 1% BSA/PBS. Samples were then immune-stained overnight (ON) at 4 °C using a rabbit anti-53BP1 antibody (Novus Biologicals, Centennial, CO, USA). After washes in 1% Bovine Serum Albumin (BSA) dissolved in PBS, samples were incubated with the anti-rabbit Alexa 488 secondary antibody (Invitrogen) for 1 h at 37 °C. Finally, slides were washed in 1% BSA/PBS, counterstained with 4,6-diamidino-2-phenylindole (DAPI; Sigma Aldrich, St. Louis, MO) and analyzed by fluorescence microscopy using an Axio-Imager Z2 microscope equipped with a coupled charged device (CCD) camera (Zeiss, Jena, Germany). The frequency of DNA damage marker foci and colocalization dots per cell were scored in 100 nuclei in at least two independent experiments.
3
Isolation and Differentiation of CD14+ Monocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CD14+ cells were isolated by negative selection from PBMCs using the Dynabead Untouched Human Monocytes kit (Invitrogen). 5 × 105 CD14 + monocytes were plated on poly-L-Lysine treated coverslips and differentiated to MDM for six days. The cells were fixed in 4% paraformaldehyde, blocked and incubated with rabbit anti-53BP1 antibody (Novus) followed by goat anti-rabbit alexa-fluor 488 second antibody. The nuclei were stained with Hoechst 333429 and the cells were visualized with a Leica SP5 confocal microscope and analyzed using ImageJ software.
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!