Alexa fluor 555 ab150078

IPEC-J2 cells were cultured in a 24-well plate. Cells were treated as described for western blot analysis. The methods used for immunofluorescence staining of cells were described previously [31 (link)] and were followed with slight modifications. In brief, after 30 min of fixation with 4% paraformaldehyde solution, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Cells were incubated with a rabbit anti-Nrf2 antibody overnight at 4 °C and were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ZF-0513, ZSGB-BIO) for 1 h. Subsequently, cell nuclei were stained with DAPI (Alexa Fluor® 555; ab150078; Abcam) for 10 min and imaged immediately using an Olympus fluorescence microscope (Tokyo, Japan). Each treatment group was analyzed in triplicate.

Immunofluorescence; rabbit anti-RAD51 PC130 1:300 (Merck Millipore Sigma Aldrich, Saint Quentin Fallavier, France), rabbit anti-geminin 52508 1:200 (CST OZYME, Saint Cyr l’Ecole, France), mouse anti-BRCA1 sc-6954 1:100 (SCBT, Heidelberg, Germany), mouse anti-γ-H2AX (H2-3F4, kind gift from Dr. Mustapha Oulad-Abdelghani, MAB-IGBMC Illkirch-Graffenstaden, 1:4000), rabbit anti-53BP1 NB100-304 1:500 (Bio-techne LTD, Abington, UK). Secondary antibodies; goat anti-mouse Alexa Fluor 488 (Abcam ab150113, 1:1000), goat anti-rabbit Alexa Fluor 555 ab150078 1:1000 (Abcam, Cambridge, UK).
Western blotting; BRCA1 9010 1:500 (CST OZYME, Saint Cyr l’Ecole, France), BRCA2 A303-434A 1:1000 (Bethyl OZYME, Saint Cyr l’Ecole, France), PARP1 WH0000142M1 1:1000 (Sigma Aldrich, Saint Quentin Fallavier, France), RAD51 8875, 1:1000 (CST OZYME, Saint Cyr l’Ecole, France) and alfa tubulin T9026 1:20000 (Sigma Aldrich, Saint Quentin Fallavier, France); secondary antibodies goat anti-mouse-HRP 70745 1:10000 (CST OZYME, Saint Cyr l’Ecole, France) and goat anti-rabbit-HRP 7076 1:10000 (CST OZYME, Saint Cyr l’Ecole, France).