Rabbit anti ncald

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).

Paraffin-embedded tissues sections were deparaffinized and rehydrated with xylene, gradient ethanol (70, 80, 95, 100%) and incubated for 20 min in 3% H₂O₂ to block endogenous peroxidase activity. After blocking with 10% goat serum and then incubated with 50 μl rabbit anti-Ki67 (Proteintech, Wuhan, China), rabbit anti-MATR3 (Proteintech, Wuhan, China), rabbit anti-NCALD (Proteintech, Wuhan, China) at 4 °C overnight. Subsequently, sections were incubated with an appropriate secondary antibody for 20 min at 37 °C and developed with diaminobenzidine and stained with hematoxylin for 5 min. After each treatment, the sections were dehydrated, cleared, mounted, and viewed under the microscope.