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J Neurosci Methods. 2000 Aug 15;101(1):9-17.

A double labeling technique using WGA-apoHRP-gold as a retrograde tracer and non-isotopic in situ hybridization histochemistry for the detection of mRNA.

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Behavioral Neurobiology Laboratory, Swiss Federal Institute of Technology, Zürich, CH-8603, Schwerzenbach, Switzerland.


We describe a novel method to study the neurochemical nature of a specific neuronal pathway by using conjugated WGA-apoHRP as a retrograde tracer and non-isotopic in situ hybridization histochemistry to examine the expression of mRNA. The technique was developed to eliminate the reduction of retrograde tracer during the rigorous procedures involved in in situ hybridization. The tracer was injected stereotaxically into the brainstem of Macaca fascicularis monkeys. Sections through the central nucleus of the amygdala were processed for the visualization of the retrogradely transported WGA-apoHRP-gold using a silver enhanced reaction, followed by non radioactive in situ hybridization for the mRNA encoding glutamic acid decarboxylase (GAD67). Numerous retrogradely labeled cells were observed in the central nucleus of the amygdala. Comparison of double-labeled sections with sections processed for the retrograde tracer alone indicated that there was relatively little loss of the retrograde tracer during the in situ hybridization processing. This method provides a relatively simple and reliable tool to study the molecular phenotype of identified projection neurons.

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