 an antibody against BrdU (No 11170376001, Hoffman-La Roche Ltd, Basel, Switzerland) that was diluted 50-fold in 0.1% bovine serum albumin/phosphate-buffered saline (BSA/PBS), revealed by CY3- conjugated donkey anti-rabbit IgG antibody, and mounted with Vectashield medium (Vector Laboratories Ltd, Burlingame, CA, USA). The numbers of BrdU-positive hepatocyte nuclei in at least five low-magnification microscopic fields of each sample (\~2000 hepatocytes) were counted.[@b23-ceg-8-175]--[@b25-ceg-8-175]

In situ hybridization
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The RNA probes were prepared. The ERα and ERβ cDNAs were cloned into pSG5 vector,[@b26-ceg-8-175] and the ^35^S-labeled antisense probes were synthesized by T7 in vitro transcription and translation system (Promega Corporation, Fitchburg, WI, USA). The specimens were prehybridized for 2 hours at 50°C in prehybridization buffer (50% formamide, 0.3 M NaCl, 10 mM Tris--HCl at pH 6.8, 10 mM NaPO~4~ at pH 6.8, 5 mM Ethylene-diaminetetraacetic aicd (EDTA), 1× Denhart's, 10 mM DTT, 500 mg/mL yeast RNA, 100 mg/mL salmon sperm DNA, and 500 nmol/mL nonlabeled α-thio-UTP \[DuPont, Wilmington, DE, USA\]). After the RNase A treatment, the slides were washed for 1 hour in the washing buffer (50% formamide, 0.3 M NaCl, 10 mM Tris--HCl at pH 6.8, 10 mM NaPO~4~ at pH 6.8, 5 mM EDTA, 1× Denhart's, and 10 mM DTT). The slides were subsequently washed in 2× Saline-Sodium Citrate buffer (SSC) for 15 minutes at room temperature, in 0.1× SSC for 15 minutes at 50°C, and then in 0.1× SSC for 30 minutes at room temperature. After dehydration of the sections, they were coated with Kodak NTB-2 emulsion, dried, and stored at 4°C. The exposure time ranged from 12 days to 15 days. Kodak D19 developer was used for 2 minutes at room temperature. The sections were then stained in toluidine blue, dehydrated in ethanol, and mounted under coverslips in Eukitt mounting medium.[@b26-ceg-8-175]--[@b29-ceg-8-175]

Reverse transcription polymerase chain reaction
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