Animals were euthanized by cervical dislocation, and the lung and heart were prepared for histopathological analysis as previously described (24 (link)). In short, excised lungs were incubated in ice-cold endotoxin-free PBS, then in 10% NBF overnight, and then in 70% industrial methylated spirit until further processing. Paraffin-embedded 4 μm sections were stained with the following antibodies: TTF-1 (ab40880, Abcam), LacZ (AB9361, Abcam), c-Jun (610326, BD Biosciences), phospho–c-Jun Ser63 (9261, NEB), phospho–c-Jun Ser73 (9164, NEB), JunD (sc74, Santa Cruz Biotechnology Inc.), HMGA2 (8179, Cell Signaling Technology [CST]), and Ki67 (M7248, DAKO).
For quantification of tumor burden, the area (μm2) of existing individual tumors in each lobe was measured with QuPath software (40 (link)) (Measure > Show Annotation Measurements Tool) and represented as a percentage of tumor area per lobe. The analysis was performed uniformity across all lung sections, and the whole lungs were used to derive data. For quantification of cell proliferation, the number of Ki67+ cells was automatically counted in individual tumors with QuPath software (Cell Analysis > Positive Cell Detection Tool) and represented as a percentage of total cells in tumor area.
For quantification of tumor burden, the area (μm2) of existing individual tumors in each lobe was measured with QuPath software (40 (link)) (Measure > Show Annotation Measurements Tool) and represented as a percentage of tumor area per lobe. The analysis was performed uniformity across all lung sections, and the whole lungs were used to derive data. For quantification of cell proliferation, the number of Ki67+ cells was automatically counted in individual tumors with QuPath software (Cell Analysis > Positive Cell Detection Tool) and represented as a percentage of total cells in tumor area.
Free full text: Click here
