In Situ Hybridization for miR-7 Detection

To evaluate the cellular distribution of miR-7 in the tumors, in situ hybridization was performed according to our previous description⁵⁶ (link) with some modifications. Briefly, before hybridization incubation, all solutions were prepared with diethyl pyrocarbonate-treated water. After deparaffinization and rehydration, tissue sections were treated by proteinase K digestion. After blocking with normal goat serum (1:100), sections were next incubated or microwave heating and then incubated with hybridization cocktail containing miR-7 probe (1:1,000 dilution; EXIQON; no. 38485-01) at 42°C for 16 hr. Then, alkaline phosphatase-labeled anti-digoxigenin antibody (1:500) (Roche Diagnostics) and the reaction products were colorized with nitro blue tetrazolium/5-bromo-4-choloro-3-indolyl phosphate (NBT/BCIP) (ZSGB-Bio). Then, the tissues were counterstained with Mayer’s hematoxylin and systematically viewed under a light microscope (Olympus IX-71).