Immunohistochemical Analysis of 8-oxo-dG

We used sorted, fixed, embedded and sectioned ISCs for 8-oxo-dG staining, a known marker of oxidative DNA damage. Sections were deparaffinized, sequentially rehydrated, and treated with a primary antibody specific to 8-oxo-dG (Trevigen, Gaithersburg, MD) according to a protocol described previously [15 (link)]. Signals were detected using DAB substrate provided in the Mouse and Rabbit Specific HRP/DAB IHC Detection Kit - Micro-polymer (Cat# ab236466, Abcam, Cambridge, MA) according to the manufacturer’s instruction. Sections were counterstained with hematoxylin, sequentially dehydrated, and mounted using Permount mounting medium (Thermo Fisher Scientific). Mounted slides were visualized under a bright field microscope and images were captured. Images were quantified using ImageJ v1.51 and the error bar represents SEM.