Sc 25304

Formalin-fixed liver was vacuum infiltrated with paraffin using a Tissue-Tek VIP 2000 and embedded with the HistoCentre III embedding station (Thermo Fisher, Waltham, MA). A Reichert Jung 2030 rotary microtome (Reichert, Depew NY) was used to prepare 4–5 µm liver sections that were stained with hematoxylin and eosin (H&E). Histological severity scoring of H&E stained liver sections was performed by a certified veterinary pathologist using the following criteria: 1, minimal, less than 25% of tissue; 2, mild, 25% to less than 50%; 3, moderate, 50% to less than 75%; 4, marked, 75–100%. Immunohistochemistry staining for CYP1A1 (sc-25304, 1:50; Santa Cruz Biotechnology, Dallas, TX) was also performed using liver sections from the paraffin-embedded tissue via TRIS/EDTA retrieval at pH 9.0. All paraffin-embedding and staining was performed by the Michigan State University Investigative Histopathology Laboratory.

The immunohistochemical analysis evaluated the expression of AHR and its downstream genes. Four-micron-thick paraffin slides were dewaxed with xylene for 30 min and then rehydrated using graded ethanol concentrations. Endogenous peroxidase was blocked with 3% hydrogen peroxide (Maixin, Fuzhou, China) for 10 min at room temperature. For the antigen retrieval procedure, we heated the slides at 98–99 °C for 15 min in a pressure cooker. All slides were then incubated with goat serum (Maixin, Fuzhou, China) for 20 min to reduce nonspecific staining. Then, the slides were incubated at 4 °C overnight with primary antibody, such as rabbit anti-human AHR polyclonal antibody at 1:100 dilution (sc-5579; Santa Cruz Biotechnology, Shanghai, China), mouse anti-human CYP1A1 monoclonal antibody at 1:50 dilution (sc-25,304; Santa Cruz Biotechnology, Shanghai, China), rabbit anti-human EGFR monoclonal antibody (SP111; Maixin, Fuzhou, China) and rabbit anti-human Ki-67 monoclonal antibody (SP6; Maixin, Fuzhou, China). Biotinylated anti-mouse and anti-rabbit antibodies (Maixin, Fuzhou, China) were applied for 15 min in a humidified chamber at room temperature. Finally, a DAB Kit (Maixin, Fuzhou, China) was used for the final chromogen analysis. PBS was used as the negative control.