Mir 21 probes

In situ hybridization (ISH) was completed on 4-μM-thick formalin-fixed paraffin-embedded kidney sections using 40 nM 5′- and 3′-DIG–labeled miR-21 probes or U6 (positive control) (Exiqon, Copenhagen, Denmark). The ISH was performed according to the manufacturer's instructions with minor modification. Deparaffinized sections were exposed to a 10-minute proteinase K (20 mg/mL) treatment at 37°C, dehydrated through ethanol solutions and then covered with hybridization buffer at room temperature for 30 minutes. The probe was diluted in hybridization buffer (40 nM, 50 μL/tissue section) and preheated at 90°C for 5 minutes to linearize. The probe was then added to the slides and incubated at 53°C for 2 hours. Slides were rinsed 2× 5 minutes in 5× saline sodium citrate (SSC), 1× SSC and 0.1× SSC at 53°C, followed by phosphate buffered saline with tween 20 washes and then blocked for 30 minutes followed by incubation with 1:800 antidigoxigenin alkaline phosphatase antibody for 1 hour at room temperature. Slides were incubated in NBT/BCIP (Roche) diluted in double-distilled H₂O for 2 hours at 34°C and then rinsed with AP stop solution, to stop the color development, before dehydration, clearing, and mounting. The tissue was photographed and analyzed.

In brief, the livers sections were treated with acetylation solution and proteinase K. Then, the sections were blocked with hybridization solution and incubated with digoxigenin-conjugated mir-21 probes (Exiqon, Denmark). Then, the sections were incubated with the HRP-conjugated anti-digoxigenin antibody (Roche, Shanghai, China). Finally, the sections were treated with NBT/BCIP (Roche, Shanghai, China). Light blue cytoplasmic staining means positive.