A SINGLE PROTOCOL TO DETECT TRANSCRIPTS OF VARIOUS TYPES AND EXPRESSION LEVELS IN NEURAL TISSUE AND CULTURED-CELLS - IN-SITU HYBRIDIZATION USING DIGOXIGENIN-LABELED CRNA PROBES

We have developed a simple non-radioactive in situ hybridization proce dure for tissue sections and cultured cells using digoxigenin-labelled cRNA probes. This protocol can be applied for the detection of variou s transcripts present at a wide range of expression levels in the cent ral nervous system. Cerebellar hybridization signals for transcripts e stimated to be expressed at high (MBP, myelin basic protein), moderate (GluR1, subunit of AMPA/kainate sensitive glutamate receptors) and lo w (inositol polyphosphate-5-phosphatase) levels of abundance are demon strated as examples. The sensitivity and cellular resolution were sign ificantly improved by avoiding any ethanol treatment commonly used in other procedures. The localization of a labelled cell with respect to its environment is shown to be more easily assessed by counterstaining of the tissue with the nuclear dye Hoechst 33258. The present protoco l can be combined with immunocytochemistry as demonstrated for glial f ibrillary acidic protein (GFAP). All steps of the procedure, including preparation and labelling of the cRNA probes, pretreatment of tissue, hybridization and visualization of the labelled transcripts, are desc ribed in detail.