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A fluorescence-based double retrograde tracer strategy for charting central neuronal connections

Nature Protocols volume 2, pages 18621868 (2007) | Download Citation



Microspheres (beads) tagged with different fluorescent markers can be used for double retrograde axonal tracing of CNS connections. They have several advantages over other double tracer techniques, including ease-of-use, high transport efficiency, distinctive cell labeling and the ability to produce well-defined injection sites. In this protocol we describe the basic procedure for their use, some common problems and how these can be overcome. The protocol, including animal surgery, preparation and delivery of tracer can be completed in approximately 0.5 d. Subsequent histological processing (excluding survival time) can be completed in 0.5–1 d.

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The authors gratefully acknowledge the supreme technical expertise of Rachel Bissett, and also many thanks to Charli Flavell for providing the photomicrographs. This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC) and the Wellcome Trust.

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  1. Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.

    • Richard Apps
  2. Department of Neuroscience, Erasmus Medical Centre, Rotterdam, The Netherlands.

    • Tom J H Ruigrok


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The authors declare no competing financial interests.

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Correspondence to Richard Apps.

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