Fitc conjugated goat anti rabbit antibody

Cells were treated with 0.3 μM AZD1775 in the presence or absence of 20 μM olaparib. For apoptosis analysis, cells were collected, washed in PBS and double-stained using an Annexin V-Phycoerythrin (PE)/7-amino-actinomycin (7-AAD) apoptosis detection kit (BD Biosciences, Erembodegem, Belgium) following the vendor’s protocol. For cell cycle analysis, cells were digested with trypsin, washed in PBS, fixed in 70% immediately prepared precooled ethanol overnight at 4 °C. After PBS washing, cells were stained with a propidium iodide (PI)/RNase staining buffer (BD Biosciences) at room temperature for 15 min in the dark according to the manufacturer’s instruction. For quantifying pHH3 and γH2AX positive cells at separate cell cycle stages, cells were fixed in 70% ice-cold ethanol overnight at 4 °C, permeabilized with 0.5% Triton X-100 (Amresco, Solon, OH) for 10 min at room temperature, incubated with primary antibodies against pHH3 (1:300) and γH2AX (1:200), probed with the FITC-conjugated goat anti-rabbit antibody (1:200; ZSGB-BIO, Beijing, China) and stained with a PI/RNase staining buffer (BD Biosciences) as described in protocols. All steps were followed by PBS washing. Samples were detected by flow cytometry within 1 h (BD Biosciences) and analyzed using FlowJo Version 7.6.1 software (FlowJo, Ashland, OR) or ModFit Version 3.0 software (Verity Software House, Topsham, ME).

The cell coverslips were placed in a 24-well plate, seeded transfected MDA-MB-231 at a density of 1.5 × 10⁵ cells per well, and incubated overnight at 37 °C. Then, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and blocked with 10% goat serum for 30 min. The cells were incubated with rabbit anti-NCALD (Proteintech, Wuhan, China) at 4 °C overnight. Cells were washed three times in PBS and incubated for 1 h at room temperature with FITC conjugated-goat anti-rabbit antibody (ZSGB-BIO, Beijing, China). After several washes, the cells were added hoechst 3342 (Beyotime, Suzhou, China) fluorescent dye solution, and coverslips were mounted to the slides using fluorescent mounting medium (PROLONG-GOLD, Thermo Fisher Scientific, MA, USA). Coverslips were imaged on the Nikon Eclipse Ti microscope (Nikon, Tokyo, Japan).