Ish buffer

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.

The thickness of the tumor and normal tissue section was 4 μm, which was treated with 20 ug/mL protease K at 37 °C for 8 min post dewaxing. The section was pre-hybridized with ISH buffer (Exiqon), followed by hybridization with digoxigenin labelled probe for 40 min at 45 °C. Hybridized sections were incubated overnight with digoxigenin antibody (Roche Diagnostics IN) at 4°C and then were stained with nitroblue tetrazole/5-bromo-4-chloro-3- indolyl phosphate.

The expression of miRNA-21, miRNA-200a and U6 snRNA was determined using miRCURY LNA™ miRNA kits (see above). Each probe was independently analyzed. After dehydration, the slides were air-dried and incubated with 40 μl of a diluted solution (1:600) of the corresponding LNA miRNA probe, which was pre-denatured by heating at 90°C for 4 min in 1 × ISH buffer (Exiqon) and hybridized at 58°C in a humidified chamber for 1 h after sealing the samples with fixogum. Following hybridization and the removal of the fixogum, the slides were washed with 5×, 1× and 0.2× SSC (5 min each wash) at 56°C. The samples were then incubated for 15 min in a blocking solution (0.1% Tween, 2% sheep serum and 1% BSA in 1× PBS) followed by incubation for 15 min in a solution containing both a FITC-anti-cytokeratin antibody (clone: CK3-6H5; Miltenyi Biotec) and an anti-DIG alkaline phosphatase antibody (Roche Diagnostics, Germany). After incubation with both of the antibodies, the samples were washed with 1× PBS for 5 min. Enzyme-labeled fluorescence (ELF) signal amplification was achieved by applying SIGMAFAST™ FastRed TR/Naphthol (Sigma-Aldrich, UK), diluted in Tris-HCl buffer according to the manufacturer's recommendations, as a substrate for alkaline phosphatase. Finally, VECTASHIELD mounting medium with DAPI (Vector Labs, Burlingame, CA, USA) was used to mount the slides.