After deposition on slides using a Cytospin centrifuge, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton in phosphate-buffered saline (PBS) and saturated with 5% bovine milk in PBS. The rabbit anti-53BP1 antibody (clone NB100-304, Novus Biologicals, Cambridge, UK) and the mouse anti-γH2AX (Ser139) antibody (clone JBW301, Merck Millipore, Darmstadt, Germany) were diluted 1/300 and 1/100, respectively, in 5% bovine milk in PBS, and deposited on cytospins for 90 min at room temperature. Slides were washed twice and antibodies to rabbit or mouse immunoglobulins conjugated to alexa 488 (diluted 1/500 in 5% bovine milk in PBS) were added for 45 min at room temperature. Slides were washed and mounted with Vectashield and 1% DAPI. Images and fluorescence were captured with a ZEISS Axio Imager Z2 microscope (× 63 objective), analyzed with Metafer (version3.6, company, town state) and ImageJ software. The number of 53BP1 and γH2AX foci was counted in at least 300 nuclei.
Supplementary informations concerning methodology are included in Supplementary experiment procedures .
Rabbit anti 53bp1 antibody clone nb100 304
Manufactured by Novus Biologicals
Sourced in United Kingdom
Rabbit anti-53BP1 antibody (clone NB100-304) is a primary antibody that specifically recognizes the 53BP1 protein. 53BP1 is a key regulator of the DNA damage response pathway. The antibody can be used in various applications to detect and study the 53BP1 protein.
Automatically generated - may contain errors
2 protocols using rabbit anti 53bp1 antibody clone nb100 304
1
Immunofluorescence Detection of DNA Damage Markers
2
Quantification of DNA Damage Foci by Immunofluorescence
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After deposition on slides using a Cytospin centrifuge (Thermo Fisher Scientific, Waltham, MA, USA), cells were fixed with 4% PFA, permeabilized with 0.5% Triton in PBS and saturated with 5% bovine milk in PBS. The rabbit anti-53BP1 antibody (clone NB100-304, Novus Biologicals, Cambridge, UK) was diluted 1/300 and deposited on cytospins for 90 min at room temperature. Slides were washed twice and secondary anti rabbit IgG conjugated to alexa 488 (diluted 1/500; Invitrogen, Life Technologies) was added for 45 min at room temperature. Slides were washed and mounted with Vectashield and 1% DAPI. Images and fluorescence were captured with an Axio Imager Z2 microscope (×63 objective, Zeiss, Oberkochen, Germany), analyzed with Metafer (version 3.6, Altlussheeim, Germany) and ImageJ softwares (National Institutes of Health, Bethesda, MD, USA). The number of 53BP1 and γH2AX foci was counted in at least 300 nuclei.
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