Rabbit anti ahr

Immunohistochemistry was conducted in 10% formalin-fixed, paraffin-embedded tissues. Dissected tissues were cleansed twice with distilled water, and residual fixatives were removed by 1 hour treatment with 1% sodium. The tissues were pre-treated with 3% hydrogen peroxide for 10 minutes, cleansed with distilled water; and cultured for 5 minutes with 1 TBST (Tris-buffered 5 saline with 0.1% Tween 20). To prevent nonspecific reactions, tissues were treated with normal goat serum (Vector Laboratories, Burlingame, CA, USA) at room temperature for 1 hour, and then cultured overnight with rabbit anti-AHR (1:300; Abcam, Cambridge, MA, USA), rabbit anti-CYP1A1 (1:300; Abcam), and rabbit anti-LC3 (1:300; Novus Biologicals, Littleton, CO, USA). Tissues were cleansed with 1 TBST, and cultured with biotinylated secondary antibody solution from the Dako REAL EnVision Detection System (Dako, Glostrup, Denmark) for 30 minutes at room temperature. The tissues then then cleansed with distilled water; counterstained with hematoxylin (Sigma-Aldrich, St. Louis, MO, USA); dehydrated and clarified based on a conventional method; and then prepared for Leica microsystem DFi8 LASX software light microscopy (Leica, Wetzlar, Germany).

We conducted the BCA assay (Sigma-Aldrich, St. Louis, MO, USA) to measure protein concentrations. Equal amounts of protein (20 µg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes (GE Healthcare, Little Chalfont, UK), and blocked with 5% skim milk in TBST for 1 h. The membranes were then incubated overnight at 4°C with rabbit anti-K10 (1:1000; Abcam, Cambridge, UK), rabbit anti-S100A7 (1:1000; Aviva Systems Biology, San Diego, CA, USA), rabbit anti-S100A8 (1:1000; LS Bio, Washington, WA, USA), rabbit anti-FLG (1:1000; LS Bio), and rabbit anti-AHR (1:1000; Abcam). Primary antibodies were detected with horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibodies (1:1000; Abcam). We visualized protein bands via enhanced chemiluminescence using a LuminoGraph II (Atto, Tokyo, Japan), and densitometric analysis of the blots was performed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).