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Fig. 7. (A) Phase-contrast photographs of CGCs after BrdU immunocytochemistry. Arrows point to BrdU labeled cells. (B) L-NAME and Shh stimulate BrdU incorporation in CGC cultures: quantification of BrdU labeled cells. Cells from neonatal rat cerebellum were cultured for 1 day in control medium or supplemented with 500 µM L-NAME or 3 µg ml^-1 Shh. Cultures were pulsed with 10 µM BrdU for the final 4 hours and processed for BrdU immunocytochemistry and subsequent quantification. Bars are the mean±s.e.m. of four experiments. ^*, P<0.01 compared with control (Bonferroni's test after ANOVA). (C) Semiquantitative RT-PCR analysis was performed with RNA isolated from CGCs treated with L-NAME or Shh for 24 hours. Amplification of the 500 bp PCR product represents N-Myc mRNA. Bars are the mean±s.e.m. of four experiments. ^*, P<0.01 (Bonferroni's test after ANOVA). (D) Double-immunofluorescence cytochemistry with anti-BrdU and anti-GFAP antibodies of CGCs cultured for 1 day in control medium or supplemented with 500 µM L-NAME. Images of immunoreactivity (left) for BrdU (green), GFAP (red) and both markers (yellow) in CGC cultures. The ratio of total BrdU-labeled cells on BrdU+GFAP-positive cells was quantified in control and compared with L-NAME-treated cultures (right). Bars are the mean±s.e.m. of three experiments. ^*, P<0.01 (Bonferroni's test after ANOVA).

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