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Fig. 6. Long-term followup of Apc-inactivated hepatocytes. (A) Survey of Apc^–/– persistence. D0, day of AdCre injection. D6, 6 days; M3, 3 months; M8, 8 months; M9, 9 months after AdCre injection. (B) Time-course PCR analysis of the presence of the ApcD ^ex14 allele. (C) Time-course GS immunostaining of Apc^–/– hepatocytes. (Scale bars, 100 m m.) (D) Protocol for tumorigenesis study.

Antibodies. Monoclonal antibodies specific for each mouse b -catenin (1/500, clone 14; BD Biosciences), mouse GS (1/200; BD Biosciences), mouse GLT1 (1/100; M. Watanabe, Hokkaido University School of Medicine, Sapporo, Japan), and mouse LECT2 (1/100; S. Yamagoe, National Institute of Infectious Diseases, Tokyo) were used as primary antibodies. Rabbit polyclonal antibodies directed against mouse Ki67 (1/400; NovoCastra, Newcastle, U.K.) and against p53 (Ab 34.1, 1/500; C. Caron de Fromontel, Institut National de la Santé et de la Recherche Médicale U590, Lyon, France) also were used.

DNA Analysis of H-Ras Mutations. Genomic DNA was extracted from microdissected nodules on 8-m m hematoxylin/eosin-stained paraffin sections with a QIAamp DNA mini kit (Qiagen, Valencia, CA), following the manufacturer’s instructions. PCRs were performed on 1/10th of the extraction product, with primers encompassing either codon 12 or codon 61 of the H-Ras gene as described in ref. 1, and were sequenced.

1. Buchmann, A., Bauer-Hofmann, R., Mahr, J., Drinkwater, N. R., Luz, A. & Schwarz, M. (1991) Proc. Natl. Acad. Sci. USA 88, 911-915.