Tissue collection and immunohistochemistry

Mice were euthanized by intraperitoneal administration of an overdose of Avertin at the time points indicated. Lungs were inflated and perfused through the trachea with 4% paraformaldehyde (PFA), fixed overnight, transferred to 50% ethanol and subsequently embedded in paraffin. Sections were cut and stained with H&E by the UTSW Histology Core, which also provided assistance with the histopathological examination. For immunohistochemistry (IHC) staining, slides were de-paraffinized and treated with Antigen Unmasking Solution (Vector laboratory, H-3300), blocked with BLOXALL™ Blocking Solution (Vector lab, SP-6000), and then incubated with anti-Ki67 (CST #9027, 1:400) or anti-pERK (CST #9101S, 1:200) at 4°C overnight. After washing extensively, slides were incubated with SignalStain® Boost Detection Reagent from Cell Signaling Technology (Rabbit #8114) at room temperature (RT) for 30 minutes. The signal was developed with ImmPACT™ DAB Substrate (Vector lab, SK-4105), and sections were counterstained with hematoxylin (Vector lab, H-3404), and mounted with VectaMount Mounting Reagent (Vector lab, H-5000). All pictures were obtained using a Zeiss microscope (Observer.Z1) with an Axiocam-MRC camera.

Ki67 and pERK1/2 index – Ki67-positive and pERK1/2 staining was distinguished by counting brown nuclei and hematoxylin (blue) counterstain. The Ki67 and pERK1/2 index for each mouse was calculated as follows: Percent positive cells = number of positive nuclei/total cell nuclei x 100, as described previously (20). For Ki67 quantification, n=2–4 animals/group were analyzed, and up to 10 independent fields/animal were quantified. For pERK1/2 quantification, 2–3 animals/group were analyzed, and 6–10 independent fields were quantified/animal.