LNA-modified, digoxigenin-labeled DNA probes complementary to human miR-146a (Exiqon) were designed for in situ hybridization of mouse and human skin sections according to the manufacturer’s instructions. A scrambled miRNA probe was a negative control, and the probe complementary to U6 snRNA served as a positive control. All the sections were deparaffinized in xylene and rehydrated in a graded series of ethanol solutions. For reagent penetration, the sections were subjected to proteinase K digestion (5–10 μg/mL) for 5 minutes at 37°C, followed by treatment with 4% paraformaldehyde in PBS for 15 minutes, and with a prehybridization solution (Biochain) at 50°C for 3–4 hours. The tissue samples were hybridized with specific probes overnight at 53°C. After the sections were washed with an SSC buffer (Biochain) dilution series, immunological detection with an alkaline phosphatase–conjugated anti-digoxigenin antibody (Biochain) was carried out. Detection of signals was based on a reaction with nitro blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate substrate (Biochain), followed by counterstaining of nuclei with nuclear fast red (Merck Millipore).

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