Fluorescent In Situ Hybridization for miR-325-3p

Constructed biotin-labeled miR-325-3p (Sangon Biotech, China) for fluorescent miRNA in situ hybridization was applied as previously described [30 (link)]. Briefly, after decalcification, cryo-cross-sectioned sections were cut into 10-μm thick slides. Then, after dewaxing and rehydrating, the slides were incubated with proteinase K (20 μg/ml, Ambion, USA), followed by prehybridization for 1 h under 37 °C temperature. After removing the prehybridization buffer, the miR-325-3p-biotin probe (5′-UUGAUAGGAGGUGCUCAAUAAA-3′-biotin) was incubated at 60 °C overnight. The following day the slides were incubated with a blocking agent (3% BSA in 0.1% PBST) for 1 h at room temperature after washing with 2 × SSC, 1 × SSC, and 0.5 × SSC, sequentially. Then the slides were incubated with Cy3-conjugated streptavidin (Jackson ImmunoResearch, USA) for 1 h. DAPI was used to label the cell nucleus. Images were captured using an Axio Imager one microscope (Zeiss, Oberkochen, Germany). For quantitative analysis, six independent sections in each group were chosen to evaluate the relative miR-325-3p positive cell ratio using Image J software.