Ab214050

Cells were fixed using 4% paraformaldehyde and lung tissues were fixed and embedded with paraffin, and incubated with antibodies against OPN (ab214050; Abcam) and Prosurfactant Protein C (ab90716; Abcam), followed by staining with fluorescein‐linked anti‐rabbit antibody (A0516; Beyotime). The cell nuclei were stained with DAPI.
The expression of OPN proteins was detected and measured by immunohistology. Sections were incubated with the primary antibody (ab214050; Abcam) at 4°C overnight and covered with the corresponding secondary antibody (ab205718; Abcam). Sections were stained with diaminobenzidine (DAB) and counterstained with hematoxylin. The ratio of integral optical density (IOD) of OPN in the area was Mean Density.²¹ Images were analyzed with Aipathwell.

Collect the correspondingly processed lung cancer cells in an EP tube, add an appropriate amount of RIPA lysate to lyse, centrifuge, and take the supernatant. Use the Bicinchoninic Acid Protein Assay kit (BCA, Solarbio, Beijing, China) to determine the total protein concentration. After adding protein buffer, bathe in boiling water for 10 min. Take an appropriate amount of total protein in 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to separate the protein, and then transfer the protein to a polyvinylidene fluoride membrane (PVDF) And sealed with 5% skimmed milk powder. Use the appropriate concentration of primary antibody to incubate overnight at 4 °C: anti-SPP1 (ab214050, 1:1000, Abcam), anti-COL11A1 (ab64883, 1:1000, Abcam), anti-E-cadherin, 1:1000 (3195S, Cell Signaling); anti-N-cadherin, 1:1000 (13116 T, Cell Signaling); anti-Vimentin, 1:1000 (5741S, Cell Signaling), GAPDH (ab8245, 1:1000, Abcam). Then, they were incubated with the appropriate concentration of secondary antibodies of the same species for 1 h at room temperature. After cleaning the membrane 3 times with 1XTBST, the protein bands were detected with an ECL luminescence reagent (Yuhengbio, Suzhou, China) and gel imaging analyzer (Bio-Rad, California, USA). Quantitative analysis of relative protein levels was performed with Quantity One software (Bio-Rad, California, USA).