Scramble mir probe

Aortic and MRA sections were de-paraffinized with xylene, followed by Proteinase K treatment (10 μg/mL for 5 min). ISH buffer (Exiqon, Vedbaek, Denmark; production #90000) was added with miR-204 probe (Exiqon, 5′-Dig-N-AGG CAT AGG ATG ACA AAG GGA A-N-Dig-3′) or with a scramble-miR probe (Exiqon, 5′-Dig-N-GTG TAA CAC GTC TAT ACG CCC A-N-Dig-3′) at 20 nM or 40 nM, and incubated for 72 h at 56 °C. After washing, the vessel sections were incubated in blocking solution for 15 min (5 mL PBS + 50 mg BSA + 100 uL Sheep serum + 2.5 uL Tween 20), followed by incubation with anti-DIG-FAB overnight (1:800 in antibody dilution solution). Slides were dipped in a solution containing BCIP/NBT (Roche, Mannheim, Germany) and incubated at 30 °C for 48 h. The slides were mounted with DPX and observed under the microscope.

In situ hybridization was performed as described [25] (link). Hybridization was done using the 5′-digoxigenin–labeled miRCURY LNA microRNA Detection Probes anti-gga-miR-16 and anti-gga-miR-15a (EXIQON, Vedbaek, Denmark). The 5′-digoxigenin–labeled Scramble-miR Probe (EXIQON) was used as a negative control.

For quantification of microvessel density (MVD) in NPC tumor samples, the number of blood vessels staining positive for CD31 (1:100 dilution, Abcam, Cambridge, MA, USA) was recorded in ten random fields at 200 magnification. The expression of MDK in NPC tumor samples was examined with IHC as previously described [16 (link)]. The antibody was purchased from Abcam (rabbit monoclonal anti-Midkine, Abcam). In situ detection of miR-9 on formalin-fixed paraffin-embedded samples was essentially performed with a miRCURY LNA™ miR-9 detection probe, using an ISH optimization kit (Exiqon, Vedbaek, Denmark) [10 (link)]. A scramble-miR probe (Exiqon) was performed as negative control.