Polyclonal rabbit anti gfp

The 3 μm paraffin-embedded sections were prepared from fixed livers and used for immunostaining of EGFP and AKR1A1. Briefly, after the sections were deparaffinized and rehydrated, they were incubated in retrieval buffer (10 mM sodium citrate, 0.05% NP-40, pH 6.0) at 100 °C for 30 min. The sections were blocked with horse serum and incubated with polyclonal rabbit anti-GFP (1:2000; GeneTex, Hsinchu, Taiwan) or polyclonal rabbit anti-AKR1A1 (1:500; Sigma-Aldrich, St. Louis, MO, USA) at 4 °C for 16 h. The sections were then incubated with biotinylated secondary antibody for 30 min, and the signal was amplified by the Elite ABC Kit (Vector Laboratories, Burlingame, CA, USA). Finally, the sections were stained with 3,3′ diaminobenzidine (DAB) and counterstained with hematoxylin. All slide images were observed and captured with a Zeiss Axio microscope (Zeiss, Germany)45 (link).

The total protein from 5 mg of liver was extracted by RIPA buffer. Twenty micrograms of the total protein was then separated by 12% SDS-PAGE and transferred to a polyvinylidene difluoride (PVDF) membrane. The membrane was blocked with 5% BSA and immunoblotted with polyclonal rabbit anti-GFP (1:4000; GeneTex) and monoclonal mouse anti-β-actin (1:500; Novus Biologicals, Littleton, CO, USA) antibodies for 16 h at 4 °C. After washing, the membrane was incubated with the horseradish peroxidase-conjugated secondary antibody for 1 h at 25 °C, and the protein bands were detected and quantified by enhanced chemiluminescence (PerkinElmer, Waltham, MA, USA) and the ImageQuant LAS 4000 mini system (GE Healthcare Biosciences, Pittsburgh, PA, USA).