Immunohistochemistry in mouse liver specimens

Liver tissue from euthanized mice was fixed in 4% paraformaldehyde for 48 hours, embedded in paraffin, and sectioned into 3–5 μm slices. Paraformaldehyde-fixed, paraffin-embedded mouse tumor and adjacent liver sections were deparaffinized, hydrated and incubated with primary antibody overnight at 4°C. Sections were stained with antibody for SOX9 (ab5535; 1:2000) from Millipore (Billerica, MA, USA). Primary antibody was detected with HRP-conjugated secondary antibody and diaminobenzidine (Dako, Carpentaria, CA, USA). Liver tissue sections were counterstained with hematoxylin.