Mouse anti human seh a 5 monoclonal antibody

Immunohistochemistry for sEH, cytokeratin 7 (CK7), and CD68 was performed as previously described [20 (link)]. Briefly, after quenching endogenous peroxidase activity and blocking nonspecific binding, sections were reacted with mouse anti-human sEH (A-5) monoclonal antibody (1 : 10 dilution; catalog no. SC-166961; Santa Cruz Biotechnology, Inc. Dallas, TX, USA), anti-human CK7 monoclonal antibody (1 : 1000 dilution, clone OV-TL 12/30, Dako Omnis, Agilent, Santa Clara, CA, USA), or anti-human CD68 monoclonal antibody (1 : 1000 dilution, clone KP1, Dako Omnis) at 4°C overnight. Further processing for colorimetric detection was according to the instructions for the VECTASTAIN Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA) using diaminobenzidine as the peroxidase substrate. The specificity of a staining reaction was assessed in several control procedures, including omission of the primary antibody or substitution of the primary antibody with nonimmune mouse isotypic IgG. Tissue sections from C57BL/6 mice livers were used as the positive controls for sEH immunostaining [23 (link)]. Sections were viewed and photographed under a differential interference contrast microscope (Nikon Eclipse 80i, Nikon Corporation, Tokyo, Japan).

Western blotting was performed as previously detailed [24 (link)]. Fifty to 100 micrograms of cytosolic proteins was separated by 8-12% SDS-PAGE, transferred to nitrocellulose membranes, and probed with the mouse anti-human sEH (A-5) monoclonal antibody (1 : 200 dilution; catalog no. SC-166961; Santa Cruz Biotechnology, Inc.) at 4°C overnight. The relative intensities of the protein signals were normalized to the intensities of the β-actin (clone AC-15, Sigma-Aldrich) signals, and the band densities were quantified by densitometric analysis using ImageJ software (National Institutes of Health, Bethesda, MD, USA; https://rsb.info.nih.gov/ij/). Tissue homogenates from C57BL/6 mice livers were used as the positive controls [23 (link)]. The sample in the vehicle control group with the lowest sEH/β-actin ratio was used as the reference for comparison, and the data are presented as fold changes.