High-resolution in situ hybridization and TUNEL staining with free-floating brain sections.

We applied in situ hybridization and the TUNEL technique to free-floating (vibratomed) sections of embryonic and postnatal mouse CNS. Full-length cDNAs specific for oligodendrocyte- or astrocyte-specific genes were labeled with digoxigenin using the random primer method. With paraformaldehyde-fixed sections, the nonradioactive in situ hybridization method provides detection of individual, very small glial progenitor cells in embryonic development. Small, isolated cells expressing oligodendrocyte specific messages can be detected in the neuroepithelium at embryonic and postnatal stages. The technique can be completed within 3 days and is as sensitive as the radioactive method. Likewise, the TUNEL method using DAB as the chromogen on free-floating sections provides excellent resolution. These DAB-stained sections can be embedded in plastic and thin-sectioned to visualize the ultrastructure of apoptotic cells. Both in situ hybridization and TUNEL methods can be applied to the same section, the tissue embedded in plastic, and semithin sections cut. The high resolution obtained with this combined procedure makes it possible to determine whether brain cells expressing glia-specific messages are undergoing apoptosis.