Liver specimens were fixed in 4% paraformaldehyde overnight at 4°C and embedded in paraffin. Hematoxylin & Eosin (H&E) staining on 4μm liver sections was performed to characterize histopathologically liver preneoplastic and neoplastic lesions in mice. Immunohistochemistry was performed as described previously [29 (link)]. Briefly, antigen retrieval was achieved in deparaffinized sections by boiling in 10mM sodium citrate buffer (pH 6.0) for 10 min. After a blocking step with the 5% goat serum and Avidin-Biotin blocking kit (Vector Laboratories, Burlingame, CA), the slides were incubated with primary antibodies overnight at 4°C. Primary antibodies used for the experiment are as follows: p-RPS6 (4858; Cell Signaling Technology), Ki67 (RM-9106; Thermo Fisher Scientific), p-4EBP1 (2855; Cell Signaling Technology), and SLC38A1 (HPA052272; Sigma-Aldrich, St. Louis, MO). These primary antibodies were selected for the analysis since they have been extensively validated by the manufacturers for immunohistochemistry. After washes, slides were incubated in 3% H2O2 for 20 minutes to quench the endogenous peroxidase, then followed by one hour of secondary antibody incubation. Signal was detected by the Vectastain ABC Elite Kit (Vector Laboratories) and visualized by DAB (Vector Laboratories).
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