Polyclonal rabbit anti akr1a1

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 liver tissues, which were obtained from anesthetized WT or Akr1A1eGFP/eGFP mice, were immediately fixed using 10% formalin for at least 16 h. The 3 μm paraffin-embedded sections of fixed liver were used for immunostaining of the AKR1A1 protein. Briefly, the sections were deparaffinized by 100% xylene and rehydrated by a series of ethanol washes (100%, 90%, 80% and 60%), and the antigens were then retrieved using retrieval buffer (10 mM sodium citrate, 0.05% NP-40, pH 6.0) at 95 °C for 30 min. The sections were incubated with 3% H₂O₂ for 10 min to block the endogenous peroxidase activity, and horse serum for 30 min and then incubated with polyclonal rabbit anti-AKR1A1 (1:500; Sigma-Aldrich) at 4 °C for 16 h. The AKR1A1 protein signal was amplified using the Elite ABC Kit (Vector Labs., Burlingame, CA, USA). Finally, the sections were stained with 3,3’-diaminobenzidine (DAB) and counterstained with hematoxylin [48 , 49 ]. The images of the sections were observed and captured using the Zeiss Axio Scope.A1 microscope (Zeiss, Germany).