Immunofluorescence in situ hybridization (IFISH) in neurones retrogradely labelled with rhodamine latex microspheres

Abstract

The method of non-radioactive in situ hybridization was developed as an alternative to radioactive assay because of the difficulties and disadvantages of the safety measures required, extensive time required for autoradiography (especially with 3H-labelled probes) and limited cellular resolution obtained using 32P- and 35S-labelled probes. This method holds great potential for studying functional anatomy of specific neuronal pathways if it can be used in conjunction with conventional tract tracing techniques. In this article we describe a simple method by which immunofluorescence in situ hybridization (IFISH) was jointly used with rhodamine latex microspheres (RLM) to trace the origin of the thalamic cholecystokininergic input in rat. RLM is a widely used retrograde fluorescence tracer and seems ideal for IFISH because: (1) it lacks aversive effect on the hybridization and immunocytochemical reactions, (2) it is resistant to the rather harsh tissue treatment required for IFISH, and (3) both the RLM and mRNA hybrids give fluorescence signals; therefore, the extent of signal co-localization can be conveniently and more accurately verified under an epifluorescence microscope. Success of the IFISH-RLM combination is chiefly limited by the quantity and availability of mRNA signals in the tissue. In our case, we used a digoxigenin (DIG)-labelled oligonucleotide probe, which through immunological amplification significantly enhanced the sensitivity of mRNA detection.