Multiple axonal tracing: simultaneous detection of three tracers in the same section |
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Authors: | José L. Lanciego Floris G. Wouterlood Elena Erro José M. Giménez-Amaya |
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Affiliation: | (1) Departamento de Anatomía, Facultad de Medicina, Universidad de Navarra, Irunlarrea s/n, E-31080 Pamplona, Spain e-mail: jlanciego@unav.es Tel.: 34-48-425600 ext. 6235; Fax: 34-48-425649, ES;(2) Graduate School Neurosciences Amsterdam, Research Institute Neurosciences Vrije Universiteit, Department of Anatomy and Embryology, Faculty of Medicine, Vrije Universiteit, 7 van der Boechorststraat, 1081 BT Amsterdam, The Netherlands, NL |
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Abstract: | Multiple neuroanatomical tract-tracing methods are important tools for elucidating the connectivity between different populations of neurons. Evaluation of the question as to whether two specific fiber inputs converge on a particular, identified population of projection neurons requires the application of a triple-staining procedure that allows the unequivocal detection of three markers in a single section. The present report deals with a combination of tracing methods using anterogradely transported Phaseolus vulgaris leucoagglutinin and biotinylated dextran amine in conjunction with retrogradely transported Fluoro-Gold. These tracers were simultaneously detected according to a three-color paradigm, which includes the use of three different peroxidase substrates (nickel-enhanced diaminobenzidine, diaminobenzidine, and Vector®VIP), thus resulting in three distinct precipitates: black, brown, and purple. We illustrate this method by showing convergence of projections arising from neurons located in two separate basal ganglia-related nuclei onto identified thalamostriatal projection neurons. |
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Keywords: | Tract-tracing PHA-L BDA Fluoro-Gold V-VIP Peroxidase. |
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