Chromogenic in situ hybridization

CISH was performed on FFPE tissue using miRCURY LNA™ microRNA ISH optimization kit (FFPE) v1.3 (Exiqon, Vedbaek, Denmark). The manufacturer’s protocol was followed with some minor changes. Briefly, 5 μm thick paraffin sections were mounted on Superfrost™ Plus glass slides and incubated overnight at 55 °C. The slides were deparaffinised with xylene and alcohol dilutions. The slides were then washed in PBS for 4 min, digested with 15 μg/ml of Proteinase K at 37 °C for 30 min, and washed in PBS before dehydration through a series of graded alcohol. The slides were hybridized with double DIG labelled Locked Nucleic Acid™ (LNA™) (Exiqon) probes at 55 °C for 1 h (see Additional file 2, S2 Table). The probe concentrations were 80 nM for the hsa-miR-18a-5p, hsa-miR-18b-5p and scramble probe, and 2.0 nM for the positive control probe U6. After hybridization the slides were washed consecutively with 5x SSC, 1x SSC and 0.2x SSC (Sigma Aldrich, St. Louis, MO, USA) at 50 °C for a total of 30 min. Then the slides were incubated with blocking solution containing 1x Maleic acid buffer (Roche, Mannheim, Germany), 10x Blocking Solution (Roche) and 2% sheep serum (Jackson Immunoresearch, Suffolk, UK) for 15 min, before application of 1:800 dilution of sheep anti-DIG alkaline phosphatase (Roche) at 30 °C for 30 min. The slides were then washed with 1% Tween-PBS for 3 × 3 min, before they were incubated with AP substrate containing NBT/BCIP (Roche) at 30 °C for 110 min. This allowed for visualization of antibody signals by NBT-BCIP. Sections were then washed twice for 5 min in KTBT buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM KCl), before being rinsed in ultrapure water 2 × 1 min. For nuclear counterstaining, the sections were immersed in Nuclear Fast Red (Sigma Aldrich, St. Louis, MI, USA) for 3 min, and then rinsed in running tap water for 5 min. Finally, the sections were dehydrated through a series of graded alcohol, and subsequently mounted by Histokitt mounting medium (VWR, Oslo, Norway).