In Situ Hybridization of miRNA Probes

miRCURY locked nucleic acid (LNA)™ microRNA detection probes, namely, hsa-miR-143, rno-miR-31, and negative controls were purchased from Exiqon (Vedbaek, Denmark). The oligonucleotides are double DIG-labeled at the 5’- and 3’-ends. ISH was performed on 6 µm FFPE sections as previously described [26 (link)-28 (link)]. Following deparaffinization, rehydration in graded alcohol and proteinase K treatment, tissue sections were hybridized with miR-143 probe (20 nM), in hybridization buffer (Exiqon) at 57°C for 14 h in a hybridizer (Dako, Glostrup, Denmark). Following stringent washes in SSC buffers, the sections were blocked against unspecific binding of the detecting antibody, using DIG wash and blocking reagent. miRNA was localized by incubation with 4-nitro-blue tetrazolium (NBT) and 5-brom-4-chloro-3′-indolylphosphate (BCIP) (Roche, Mannheim, Germany). Nuclear fast red (Vector Lab., Burlingname, CA) was used as a counterstain.