Nonradioactive in situ Hybridization Techniques for Routinely Prepared Pathology Specimens and Cultured Cells

We report a modified method for non-radioactive in situ hybridization suitable for semiquantitative analysis of specific mRNA sequences in fixed cultured cells or formalin fixed, paraffin embedded tissue sections. Our modifications include random primer incorporation of digoxigenin-label into DNA probe; specimen treatment with proteinase K; heating of specimen and hybridization solution to 100°C for 10 min followed by hybridization at 42°C for 18 hr; and improved immunologic detection with an antibody against digoxigenin conjugate. Visual results were obtained within 3 days. Probe sizes as large as 1.8 kb resulted in good specific signals with minimal background. Specific histologic localization can be semiquantitated under a brightfield microscope linked to a computer aided densitometry system. (The J Histotechnol 17:313, 1994)