Figure 6

Verification that in situ hybridization labels specific endogenous transcripts. To verify that the in situ hybridization detects specific endogenous transcripts, average labelling of the cell bodies was quantified from more than 30 cells for each probe and compared to a non-specific (NS) RNA probe. Error bars show standard error of the means. The Student t-Test shows all probes produce much stronger labelling in the cell body than observed with the non-specific probe (P < 0.0001) which was not different than labelling seen when the RNA probe was completely omitted from an otherwise identical protocol (data not shown). The in situ procedure used for these studies was adapted from a previous study [34]and involved hybridization of a digoxigenin-labelled RNA probe, labelling of this probe with an anti-digoxigenin fluorescein-conjugated antibody followed by amplification with a secondary Cy3-conjugated mouse monoclonal anti-fluorescein antibody. All images were acquired on a Zeiss LSM 510 confocal laser scanning microscope.