We describe here a protocol developed to detect specific mRNAs by in situ hybridization using tissue sections that were not treated to inactivate RNase and were stored in cryoprotectant solution for several years. Brains from rats, monkeys and humans were sectioned at 50 μm and stored free floating in an ethylene glycol based cryoprotective solution at -20°C. Rat brain sections were kept in cryoprotective solution for 3 days, 1 month and 2 months. Control sections were cut and mounted immediately on gelatin-coated slides and stored at -80°C. Monkey brain sections were stored in cryoprotective solution for up to 5 years. Human sections were tested after storage for one year. Oligonucleotide probes that were complementary to human preproenkephalin mRNA (amino acid sequences 130–145), rat preproenkephalin mRNA (sequences 388–435) and rat tyrosine hydroxylase mRNA (sequences 1441–1488) were labeled with35S-dATP and terminal deoxynucleotidyl transferase. To prevent possible RNase contamination from mounting the tissue sections onto gelatin coated slides, the in situ hybridization was performed in sterile culture dishes. Following each step, solutions were aspirated out of the dish. The amount of probe necessary for each section was 45 μl (rat), 450 μl (monkey), and 350 μl (human). Using this protocol, the detection of specific mRNAs in rat brain sections was more specific with less non-specific background as compared to control sections that were processed after they were mounted onto gelatin-coated sides. Excellent resolution was also obtained from monkey brain sections that were stored in cryoprotectant for up to 31 months and in human brain sections stored for 12 months. Similar to conventional in situ hybridization methodology, the prehybridization treatment step was not necessary to reduce the background. Using this protocol, it is possible to obtain specific mRNA labeling with low background in brain sections that have been stored for several years in cryoprotectant solution.
