Cells were grown on glass coverslips. After at least 24 h of culture, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 and stained with primary antibodies over-night at 4 °C. 53BP1 (NB100-304) and FANCD2 (NB100-182) antibodies were purchased from Novus Biologicals, γH2AX antibody from Merck/Millipore (05–636), and RAD51 antibody from Santa Cruz biotechnology (sc-8349). Cells were washed three times with PBS Tween 0.1% and incubated with the secondary antibodies for 1 h (Rhodamine Red X (R6394) and AlexaFluor 488 Goat anti-mouse (A11017) or anti-rabbit (A11070), purchased from Invitrogen). DNA was stained with 4.6-diamino-2-phenyl indole (DAPI). Cells were counted positive for foci formation when >10 foci/nuclei were detected.
Rhodamine red x r6394
Manufactured by Thermo Fisher Scientific
Rhodamine Red X (R6394) is a fluorescent dye produced by Thermo Fisher Scientific. It is designed for use in various scientific applications that require a bright, red-emitting fluorescent label. The dye exhibits excitation and emission maxima at 575 nm and 602 nm, respectively. Rhodamine Red X can be used for labeling proteins, nucleic acids, and other biomolecules.
Automatically generated - may contain errors
2 protocols using rhodamine red x r6394
1
Immunofluorescence Staining of DNA Damage Markers
2
Immunofluorescence Assay for DNA Damage
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Cells were grown on glass coverslips. After at least 24 hours of culture, cells were fixed with 4% paraformaldehyde, permeabilized with 0.5% Triton X-100 for 15 minutes, blocked in 3% BSA and stained with primary antibodies (dilution 1:3000) overnight at 4 °C in blocking solution. For FANCD2 detection, a pre-extraction step (0.5% Triton X-100 for 5 minutes) was performed before fixation. 53BP1 (NB100-304) and FANCD2 (NB100-182) antibodies were purchased from Novus Biologicals and γH2AX antibody from Merck/Millipore (05-636). Cells were washed three times with PBS Tween 0.1% and incubated with the secondary antibodies (dilution 1:800) for 1 hour (Rhodamine Red X (R6394) and AlexaFluor 488 Goat anti-mouse (A11017) or anti-rabbit (A11070), purchased from Invitrogen). DNA was stained with DAPI. Cells were counted positive for γH2AX and 53BP1 foci formation when > 10 foci/nuclei were detected and counted positive for FANCD2 when > 3 foci/nuclei were detected.
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