New Cre/lox Genetic Tools Figure 6. Morphologies of individual AP+ type 1 DA cells from the retinas of TH-IRES-CreER;R26IAP mice. A single 100–300 mg 4HT injection was administered IP between P3 and P8; retinas were analyzed ,6 weeks later. Retina flat mounts were processed for AP histochemistry and clarified in BBBA. Axon-like processes (green) and dendrites (blue) were traced with Neuromantic software. The soma is indicated by a red arrow. The outline of the flattened retina and the location of the optic disc are shown in red. (A) Twelve retinas, each of which had a single type 1 DA cell. (B) A retina with two type 1 DA cells. (C) Image of the boxed region of the retina shown in (B). The dendrites (lower right) are uniformly thicker and more darkly stained than the axon-like processes. (D) A putative type 2 DA cell. Scale bars in B and D, 500 mm. Scale bar in B applies to panels A and B. BBBA causes ,30% tissue shrinkage. doi:10.1371/journal.pone.0007859.g006 recombination is more difficult and can require near-toxic doses of 4HT administered over multiple days [3]. By contrast, for sparse cell labeling experiments, the challenge is reversed: the problem is now to reduce the efficiency of 4HT- dependent Cre-mediated recombination and eliminate any background of 4HT-independent recombination. As shown in Figure 3A and B, when using CreER alleles with relatively high levels of expression (e.g. R26CreER) in combination with relatively efficient loxP targets (e.g. R26IAP), background recombination is substantial even in the absence of 4HT. Using a less efficient loxP target, such as Z/AP, reduces the recombination level so that there are few or no labeled cells in the absence of 4HT [10]. However, in our experience, the Z/AP locus is unusual in this respect, and most loxP target loci more closely resemble R26IAP in recombi- nation efficiency. Long and Rossi [46] have suggested that the unusually inefficient recombination efficiencies of the Z/AP and PLoS ONE | www.plosone.org 9 November 2009 | Volume 4 | Issue 11 | e7859