In Situ Hybridization Using Riboprobes on Free-Floating Brain Sections

A method is described for in situ hybridization of riboprobes to free-floating brain sections. Brain sections are hybridized and processed free-floating in buffer, i.e., without attachment to a support such as a slide. To withstand the extra wear compared with sections processed on-slide, the brain tissue must be well fixed (4% paraformaldehyde) and sections cut at thickness of typically 40 μm. Sections were exposed to a prehybridization treatment before a riboprobe is added to form the hybridization solution. Riboprobes were prepared from cDNA via an in vitro transcription reaction and are labeled with digoxigenin. The sections are subsequently processed to remove nonspecific binding and the digoxigenin label detected via an antibody conjugated to alkaline phosphatase. This method may be readily combined with neuronal tracing and is ideal for further processing by immunohistochemistry to detect specific proteins.