Immunohistochemical Analysis of Liver Tissue

Liver specimens were fixed in 10% paraformaldehyde and then embedded in paraffin. Immunohistochemistry was performed as previously described 33 (link)-35 (link). Tissue slides were deparaffinized in xylenes and rehydrated in graded ethanol and then boiled in 10 mM citrate buffer (pH 6.0) for 30 min by placement in a microwave, followed by cool down at room temperature. 3% hydrogen peroxide in methanol was used to quench endogenous peroxidase activity by incubation for 10 min. After blocking with the blocking solution (PBST/5% normal goat serum), slides were then incubated with primary antibodies overnight at 4 °C. Subsequently, tissue slides were incubated with SignalStain® Boost IHC Detection Reagent (Cell Signaling Technology, USA) for 30 min at RT. Finally, tissue sections were developed with SignalStain® DAB Substrate (Cell Signaling Technology, USA) and counterstained with hematoxylin.