Immunostaining of EGFP and AKR1A1 in Liver

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).