| Intravital administration of affinity ligands |
10 min |
Two iv injections: TAM marker 18 h before killing, lectin 1 h prior. |
|
In vivo perfusion and pulmonary resection |
20 min |
Perfusion through right ventricle with PBS followed by 4% FA ensures lung perfusion and fixation. Whole-animal perfusion also possible through left ventricle. |
| Post-perfusion fixation |
3 h |
Room temperature. Apply vacuum 1–2 × to remove air from alveoli. |
| Clearing |
24–48 h |
Performed under gentle rocking, 37 °C. Lungs prone to trapping air if shaking too rapid. |
| Mesocopic imaging of entire lung |
20 min |
Since lungs are of similar size, process is streamlined by using the same imaging grid program over multiple samples. |
| × 10 Confocal imaging of ∼1 mm tumour |
20 min |
|
| × 10 Imaging of entire lung |
Hours |
Results in extremely large file sizes. Spot imaging of tumours is a practical alternative to whole organ imaging at × 10. |