Combined fluorescent in situ hybridization and immunofluorescence: limiting factors and a substitution strategy for slide-mounted tissue sections.

The simultaneous localization of several anatomical markers is often required to understand and analyze the organization of complex brain nuclei or identify neuronal networks recruited during a specific biological stimulus. Gathering such information is usually achieved by the combined detection of both mRNA and proteins. Staining techniques using fluorescence have progressively overtaken the use of radioactive tissue labeling and immunostaining based on the avidin-biotin-peroxidase complex. Despite the promise offered by the combination of fluorescent in situ hybridization (FISH) and immunofluorescence (IF), in terms of reduced bench time and easy visualization of multiple labels at once, some technical hurdles have to be overcome to produce reliable data from these state-of-the-art neuroanatomy techniques. Here, we have adapted a combination of FISH and IF for slices mounted on a microscope slide, using mRNA (GAD65 mRNA) and proteins (NeuN, FosB or TH) widely studied in neuroanatomy, to validate this method. Proteinase K (PK), which is often used to optimize riboprobe penetration, is a major limiting factor in obtaining successful IF labeling. This study demonstrates the inaccuracy of PK and provides appropriate tools to improve the efficiency of the combined FISH-IF procedure to obtain high quality fluorescent multi-labeling.