Goat anti rabbit or anti mouse secondary antibodies

The Bradford protein assay kit (Beyotime, Shanghai, China) was used for protein concentration measurement. The anti-β-actin and anti-GAPDH antibodies were purchased from Abcam. The anti-E-cadherin, anti-N-cadherin, anti-Vimentin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The anti-Rb, anti-pRb, anti-CDC25C, anti-CDC25A, anti-p53, anti-p27, anti-CDK2, anti-CDK4, anti-CDK6, anti-cyclinD1 and anti-cyclinE1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).The goat anti-rabbit or anti-mouse secondary antibodies were bought from Affinity Biosciences (Cincinnati, OH, USA). The immunoreactive bands were visualized using the MiniBIS Pro gel imaging system (DNR, Israel).

The samples were sequentially dehydrated, embedded in paraffin and cut into 4 µm sections and then fixed in 4% paraformaldehyde solution for 48 h at room temperature. Paraffin-embedded sections of lung tissue and cells underwent antigen retrieval (110˚C; 50 kPa; 10 mmol/l citric acid-sodium buffer) for 80 sec, followed by quenching endogenous peroxidases with 3% H₂O₂ for 15 min at room temperature. Sections were incubated with primary antibodies against SPP1 (1:100; cat. no. AF0227; Affinity Biosciences) overnight at 4˚C. The following day, they were incubated with goat anti-rabbit or anti-mouse secondary antibodies (cat. no. PV-6000; Beijing Zhongshan Jinqiao Biotechnology Co. Ltd.) at 37˚C for 30 min. Color development was performed by 3,3'-diaminobenzidine (ZLI-9018; OriGene Technologies, Inc.) and brown staining was considered as positive. Sections were mounted with neutral balsam and observed by microscopy (magnification, x400; BX53; Olympus Corporation).