In Situ Hybridization of mRNA with Biotin and Digoxigenin Labeled Probes

Abstract

We have developed a reliable nonisotopic mRNA in situ hybridization (ISH) technique utilizing specific antisense riboprobe and oligodeoxynucleotide probes labeled with biotin or digoxigenin to detect human glial fibrillary acidic protein and human S-100β protein in formalin fixed, paraffin embedded human and rat brain tissue sections. This method also allows detection of T and B cell rearrangements with commercially prepared oligodeoxynucleotide probes for kappa and lambda light chains in lymphatic tissues. The method uses proteinase K digestion and overnight hybridization with the labeled probes. Detection of the hybridized probe is accomplished with either streptavidin alkaline phosphatase or with anti-digoxigenin-AP. Hybridization is visualized after enzyme reaction with nitro blue tetrazolium substrate at the light microscopy level. ISH with biotin or digoxigenin labeled oligodeoxynucleotide probes and riboprobes is a rapid, accurate and specific procedure for the study of mRNAs in tissue sections. It correlates well with results obtained by immunohistochemistry. This ISH protocol should be extendible for the detection of other mRNAs for which the sequence is known. (The J Histotechnol 17:307, 1994)

Keywords:

• biotin
• digoxigenin
• in situ hybridization
• oligodeoxynucleotide probe
• riboprobe
• RNA