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A light microscope-based double retrograde tracer strategy to chart central neuronal connections

Nature Protocols volume 2, pages 18691878 (2007) | Download Citation



This protocol describes a double retrograde tracing method to chart divergent projections in the CNS using light microscope techniques. It is based on immunohistochemical visualization of retrograde transport of cholera toxin b-subunit (CTb) and silver enhancement of a gold–lectin conjugate. Production of the gold–lectin is explained in detail, and a technique is offered to record through the injection pipettes, to help guide accurate placement of injections. Visualization of the two tracers results in light brown staining of CTb-labeled neurons and labeling by black particles of gold–lectin-containing neurons. Both types of label are easily recognized in the same neuron. The labeling is permanent and is well suited for studies in which large areas of the brain need to be surveyed. The whole procedure (excluding survival time) takes approximately 5–7 d to complete.

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The authors gratefully acknowledge the supreme technical expertise of Hans van der Burg and Erika Sabel-Goedknegt in the development of this protocol. This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC) and the Wellcome Trust (to R.A.), and by the Netherlands Organisation for Scientific Research, Division of Earth and Life Sciences and the Dutch Ministry of Health, Welfare, and Sports (to T.J.H.R.).

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  1. Department of Neuroscience, Erasmus MC Rotterdam, PO Box 2040, 3000 CA Rotterdam, The Netherlands.

    • Tom J H Ruigrok
  2. Department of Physiology, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.

    • Richard Apps


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The authors declare no competing financial interests.

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Correspondence to Tom J H Ruigrok.

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