Fitc labelled secondary antibody

For immunofluorescence staining, A549 and PC9 cells were seeded on 10 mm confocal dishes and treated with 0.1 μmol/L pemetrexed and/or 10 μmol/L ribociclib. Equal amounts of DMSO were added to control cells. After 72h, cells were fixed in 4% PFA for 30 min, permeabilized in 0.3% Triton X-100 for 20 min, and blocked in 5% normal goat serum for 60 min at room temperature. Sequentially, cells were incubated with primary antibody against CDK4 (1:200 dilution, #ab108357, Abcam, USA) or phosphor-Rb (1:200, #8516T, Cell signalling Technology, USA) at 4°C overnight, then washed with phosphate buffered-saline with Tween-20 (PBST) and incubated with FITC-labelled secondary antibody (1:100 dilution, Proteintech, Wuhan, China) for 1 h at room temperature. The nuclei were labelled using 4′,6-diamidino-2-phenylindole (DAPI) (2 mg/mL) in the dark for 15 min, and imaging was performed on a fluorescence microscope (Olympus IX 73 DP80, Japan).

Liver sections were fixed with 4% paraformaldehyde for 15 min at 37°C. Next, the sections were blocked with 2% BSA for 30 min. Subsequently, the sections were incubated with an anti-Srebp-1 antibody (1:100) overnight at 4°C. The sections were then washed and re-incubated with an FITC-labelled secondary antibody (1:100) (ProteinTech, Chicago, United States) for 1 h. Finally, immunofluorescence was observed using a fluorescence microscope after staining with DAPI for 5 min.