In situ hybridization: Use of S-35-labeled probes on paraffin tissue sections

The following protocol is for radioactive In situ hybridization detection o f RNA using paraffin-embedded tissue sections on glass microscope slides. S teps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) t reatment of solutions and baked glassware are unnecessary. The tissue is fi xed using 4% paraformaldehyde, hybridized with S-35-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2 +/-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) in corporation of label into the probe, and (3) amount of background signal. A dditional steps involved in the autoradiography process, including developm ent of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed . In addition, a general guide to the interpretation of the in situ results is provided. (C) 2001 Academic Press.