Formical 4

Samples were fixed in 10% neutral buffered formalin (NBF, StatLab Medical Products, Brooklyn, NY) for 24 ​h and decalcified with Formical-4 (StatLab Medical Products, Brooklyn, NY) for 15–20 days until decalcification endpoint. Paraffin blocks were sliced at 4 ​μm and stained with Safranin O-Fast Green (Sigma-Aldrich, St. Louis, MO, HT90432, F-7252) in the sagittal plane to evaluate distribution of proteoglycans in the cartilage. Slides were imaged (20x) using an EVOS FL Auto microscope (Life Technologies, Carlsbad, CA). Histological focus was kept consistent with the ROI analyzed at UHF-MRI. Histological grading was scored following Laverty et al. standards [38 (link)].

Mouse ear fibroblasts (Merfs) were derived and maintained as described previously (Stanley et al. 2016 (link)). For histopathology studies, mice were individually euthanized by exposure to gradually increasing concentrations of CO₂, then perfusion with heparinized saline followed by 10% NBF via left cardiac ventricle. Perfused tissues were fixed in 10% NBF for at least 24 h prior to trimming. The head and hindlimb were separately decalcified with Formical 4 (StatLab Medical Products). Gross examination and tissue collection was performed as described in Brayton et al. (2014) (link). Sections (∼5 µm) were stained with hematoxylin and eosin and reviewed by two veterinary pathologists (B. Kang and C. Brayton). Image capture was performed on a Nikon 55i microscope and slides were scanned at 20x on an Aperio AT2 instrument (Leica Biosystems). Immunohistochemistry was performed using standard methods on formalin-fixed paraffin-embedded tissues using these antibodies: Ki67 (SP6, Abcam, ab16667), Cleaved caspase 3 (Asp175, 5A1E, rabbit mAb #9664, Cell Signaling Technology), and ZCCHC8 (R34469, polyclonal rabbit, Sigma-Aldrich).

For this lifetime carcinogenesis study, all disease states were interpreted within the context of a systematic pathologic evaluation directed by board-certified veterinary pathologists (E.F.E. and D.A.K.). Structured necropsy and tissue collection protocols were followed for each mouse and involved photodocumentation of all gross lesions, collection of frozen tumor material, and preservation of tumor material in RNAlater. All tissues were grossly evaluated for all mice. To evaluate brain tissues and Harderian glands, craniums were decalcified for 48 hours in Formical-4 (StatLab, McKinney, TX 75069, product 1214) and five coronal sections of the skull were reviewed for each mouse. All gross lesions were evaluated microscopically and fixed in 10% neutral-buffered formalin and paraffin-embedded, and 5-μm sections were stained with hematoxylin and eosin (H&E) and evaluated by a veterinary pathologist. For mice with solid tumors, all lung fields were examined histologically to detect the presence or absence of micrometastases. Tumor nomenclature was based on consensus statements produced by the Society of Toxicologic Pathology for mouse tumors (www.toxpath.org/inhand.asp). Representative histologic images routinely stained with H&E are presented in figs. S2 (A to E) and S3 (A and B).