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Labeling and visualizing cells and subcellular structures within thick tissues, whole organs, and even intact animals is key to studying biological processes. This is particularly true for studies of neural circuits where neurons form submicron synapses but have arbors that may span millimeters in length. Traditionally, labeling is achieved by immunofluorescence; however, diffusion of antibody molecules (&gt;100 kDa) is slow and often results in uneven labeling with very poor penetration into the center of thick specimens; these limitations can be partially addressed by extending staining protocols to over a week ( Drosophila brain) and months (mice). Recently, we developed an alternative approach using genetically encoded chemical tags CLIP, SNAP, Halo, and TMP for tissue labeling; this resulted in &gt;100-fold increase in labeling speed in both mice and Drosophila , at the expense of a considerable drop in absolute sensitivity when compared to optimized immunofluorescence staining. We now present a second generation of UAS- and LexA-responsive CLIPf, SNAPf, and Halo chemical labeling reagents for flies. These multimerized tags, with translational enhancers, display up to 64-fold increase in sensitivity over first-generation reagents. In addition, we developed a suite of conditional reporters (4xSNAPf tag and CLIPf-SNAPf-Halo2) that are activated by the DNA recombinase Bxb1. Our new reporters can be used with weak and strong GAL4 and LexA drivers and enable stochastic, intersectional, and multicolor Brainbow labeling. These improvements in sensitivity and experimental versatility, while still retaining the substantial speed advantage that is a signature of chemical labeling, should significantly increase the scope of this technology.

Second-GenerationDrosophilaChemical Tags: Sensitivity, Versatility, and Speed