Hpa010136

Immunohistochemical staining with monoclonal mouse anti-human mitochondria (hMIT) (clone 113-1, Merck Millipore, Burlington, MA, 1:2000), antihuman CYP1A2 (clone 3B8C1, Abcam plc., Cambridge, UK, 1:1000), antihuman CYP2C9 (clone 2C8, LifeSpan Biosciences, Inc., Seattle WA. USA, 1:150), and rabbit antihuman CYP3A4 (clone EPR6202, Abcam Plc., 1:300) antibodies was performed as described previously^{14 (link)}. Tissues were fixed in 4% (v/v) phosphate-buffered formalin (Mildform 10 NM; Wako Pure Chemical Industries). The sections were autoclaved for 10 min in a target retrieval solution (0.1 M citrate buffer, pH 6.0; 1 mM EDTA, pH 9.0), equilibrated at room temperature for 20 min, and then incubated with an anti-POR (HPA010136, Sigma) primary antibody. Primary antibodies were visualized using amino acid polymer/peroxidase complex-labeled antibodies (Histofine Simple Stain MAX PO [MULTI]; Nichirei Biosciences Inc.) and diaminobenzidine (DAB; Dojindo Laboratories, Kumamoto, Japan) substrate (0.2 mg/mL 3,3-diaminobenzidine tetrahydrochloride in 0.05 M Tris–HCl, pH 7.6, and 0.005% H₂O₂). The sections were counterstained with hematoxylin. Images were captured using a digital slide scanner (NanoZoomer S60; Hamamatsu Photonics, KK Hamamatsu, Japan).

Liver microsomes (5–20 μg) were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electrophoretically transferred to a polyvinylidene difluoride membrane (Merck, Darmstadt, Germany). After blocking with 0.5% non-fat milk in Tris-buffered saline (50 mM Tris, 138 mM NaCl, 2.7 mM KCl) containing 0.05% Tween 20 (v/v) at room temperature for 30 min, the membrane was probed with anti-human POR antibodies (1:2000; HPA010136, Sigma), anti-human CYP1A2 antibodies (1:2000; 19936-1-AP, Proteintech, Rosemont, IL, USA), anti-human CYP2A antibodies (1:5000; PAP061, Nosan Corporation, Yokohama, Japan), anti-mouse P450 2b10 antibodies (1:500; AB9916, Merck Millipore), anti-human P450 2C9 antibodies (1:1000; HPA015066, Sigma), anti-rat P450 2D1 antibodies (1:500; BML-CR3210, Enzo Life Sciences, Farmingdale, NY, USA), anti-rat/human CYP2E1 antibodies (1:2000; CR3271, Enzo Life Sciences), anti-rat CYP3A2 antibodies (1:1000; R-PAP 171, Nosan Corporation), or anti-protein disulfide isomerase (PDI) antibodies (1:200; 11245-1-AP, Proteintech) at room temperature for 1 h. The membrane was then incubated with goat anti-rabbit IgG antibodies (1:5000; GE Healthcare, Chicago, IL, USA) at room temperature for 20 min. Reactive bands were visualized using the ECL Prime Western Blotting Detection System (GE Healthcare).