Protein diluent

Mice bearing the Eμ-myc transgene were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). The strain was maintained by breeding hemizygous Eμ-myc transgenic males with wild-type C57BL/6 females. To test the efficacy of AGN and decursin, the mice were administered AGN (200 mg/kg) for eight weeks or decursin (10 mg/kg, ChemFaces) for four weeks. After sacrificing the mice, H&E staining were performed as previously described^{47 (link)}. For the immunohistochemical (IHC) staining of Myc in the spleen tissues, a polyclonal anti-Myc primary antibody (Abcam; ab34072), suitably diluted with a protein diluent (Dako), and a polymer-horseradish peroxidase anti-rabbit (Dako) secondary antibody were used followed by 3,3- diaminobenzidine treatment to visualize the proteins. IHC and H&E-stained samples were examined at 100× magnification (scale bar, 100 μm) with an Olympus CX31 microscope (Olympus Corporation, Tokyo, Japan). The representative images were photographed using a digital photomicrographic camera attachment Moticam 2000 (Motic Co. Ltd., Kowloon, Fujian, China) and Motic Images Plus 2.0 software (Motic Co. Ltd., Kowloon, Fujian, China) and then assembled with PowerPoint software (Microsoft, Redmond, WA, U.S.A).

To examine hepatic morphology and assess liver fibrosis, H&E staining and Sirius red staining were performed, respectively. Liver specimens were fixed in 10% neutral buffered formalin (Sigma), embedded in paraffin and cut into 4 μm sections. Next, the specimens were deparaffinized, hydrated and stained by standard methods.
For immunohistochemistry, liver sections were deparaffinized, hydrated and incubated in 3% hydrogen peroxide, to block endogenous peroxidase. Antigen retrieval was performed by heating in 10 mM sodium citrate buffer (pH 6.0) for 10 min using microwave. Specimens were blocked in Protein Block solution (Dako) for 30 min at room temperature (RT) followed by incubation with primary antibody at 4 °C overnight. Other sections were also incubated at 4 °C overnight in non-immune sera. Rabbit α-SMA antibody (diluted 1:500; Abcam) was used as a primary antibody and diluted in Protein Diluent (Dako). Polymer-horseradish peroxidase anti-rabbit (Dako) was used as secondary antibody and 3,3′-diaminobenzidine as brown colour was used to visualize the protein.

Liver specimens were fixed in 10% neutral buffered formalin (NBF; Sigma), embedded in paraffin and cut into 4 µm sections. The specimens were deparaffinized, hydrated, and stained with H&E to examine hepatic morphology.
For immunohistochemistry, liver sections were deparaffinized, hydrated, and incubated in 3% hydrogen peroxide to block endogenous peroxidase. Antigen retrieval was performed by heating in 10 mM sodium citrate buffer (pH 6.0) for 10 min in a microwave. The specimens were then blocked in Protein Block solution (Dako, Carpinteria, CA, USA) for 30 min at room temperature (RT) followed by incubation with primary antibody at 4 °C overnight. Other sections were also incubated at 4 °C overnight in non-immune sera. Mouse Ki67 antibody (diluted 1:2000; Novocastra, Leica Microsystems, Newcastle upon Tyne, UK) was used as a primary antibody and diluted in Protein Diluent (Dako). Polymer-HRP anti-rabbit (Dako) was used as a secondary antibody and 3,3′-diaminobenzidine (DAB) for brown color was used to visualize the protein. To quantify Ki67-positive hepatocytic cells, 10 randomly chosen 20× fields/section were evaluated by counting the total number of Ki67-stained cells/field for each mouse.