Figure 2

CNS explants from Cre-dependent transgenic embryos displayed reporter activity upon exposure to CPP-Cre fusion proteins. Telencephalic explants of dissected fragments from the embryonic brain (see Additional file 3), were obtained and cultured as previously described, then exposed to protein conjugates added at the center of the explant (asterisk). Two to ten days later, transgene-expressing cells (arrows) were detected by reporter enzymatic activity (A, B), intrinsic fluorescence (C), or immunohistochemistry (D-E). Vehicle-treated explants were devoid of reporter expressing cells (see Additional file 4). (A, B) Explants (R26R strain) exposed for 96 h to a 1 μl drop of 100 ng/μl H3NC. Visualization of reporter activity (X-gal staining; positive blue cells indicated by arrows) showed reporter-expressing cells, located mainly in the peripheral domain (B). (C) Explant from RYFP strain, exposed for 8 h to a 1 μl drop of 100 ng/μl H3NC. Live fluorescence recording (GFP channel; bright green cells, arrow) shows early recombinant cells in the centre of the explant. (D-E), YFP immunofluorescence analysis using a pan-GFP antibody revealed reporter expression in large cell clusters, 72 h after H3NC transduction (1 μl drop of 100 ng/μl). In (D), labelled cells were classically documented while in (E), reporter YFP was analysed using confocal analysis (pseudo-colors GLOW-LUT converted representing signal intensity, blue indicating saturated signal). In A-E, arrows point to representative recombinant cells, including those with a morphology suggestive of neuronal identity in pictures D-E. For the whole figure, the scale bar in A represents: A, 350 μm; B, 750 μm; C, 150 μm; D, 50 μm; E, 1000 μm.