Double immunofluorescence for Cre and GFP using Cy3 and FITC labelled secondary antibodies respectively with single excitations of 543 nm to detect Cre (A, C) or 488 nm to detect GFP (B, D). LV-Cre-EGFP virus (A-B) and AAV-Cre/AAV-GFP mixture viruses (C-D) were injected into the hippocampus and neocortex of Rosa26 mice 4 weeks before perfusion. The patterns of expression of the two transgenes are almost identical (allowing for the slightly stronger staining obtained with the tyramide-enhanced Cre immunohistochemistry) irrespective of the vector used. Fig. 3E,3F, and 3G show the extent of colocalization of GFP and Cre in the dentate gyrus of an AAV-Cre/AAV-GFP mixture injected Rosa26 mouse brain. In the original micrographs, more cells were Cre positive: 373 cells were Cre positive and 256 cells were GFP positive. However the few green cells in 3G show that some cells expressed GFP alone. The arrowheads and arrows in A-D indicate immunofluorescence in pyramidal cells of CA1 and CA2 respectively. Scale bars, A-D = 200 μm, E-G = 400 μm.