transcriptase according to the protocol. The following primers were used for the PCR amplifications: igf-1a, 59- AGCGGT- CATTTCTTCCAG-39 and 59- CCTTACAGGAAGAGTGGC- TAT -39; igf-1b, 59- GTGCTGCGTTCTCATCCT -39 and 59- TGTTGAGGAGGTTTGGGT -39; igf-2a, 59- TAACCCTGT- CTGCCTTCG -39 and 59- CTGTGGGAAGAAGGATGG -39; igf-2b, 59- TCAAACAGCCGCCGTCCTC -39 and 59- TGG- GACGATGGCAGGTTG -39; b-actin, 59- CTTGCGGTATC- CACGAGAC -39 and 59- GCGCCATACAGAGCAGAA -39. PCR cycles and amounts of templates were optimized for each primer set in pilot experiments. PCR cycles were as follows: 95uC for 3 min, 34 cycles of 94uC for 30 s, 53uC (igf-1a and igf-1b) or 56uC (igf-2a, igf-2b, and b-actin) for 30 s, and 72uC for 1 min. PCR products were analyzed by electrophoresis followed by ethidium bromide staining and a no-template control was the negative control. Whole mount in situ hybridization using digoxigenin (DIG)-labeled RNA riboprobe was carried out essentially as reported previously [63]. 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