5 brom 4 chloro 3 indolyl phosphate bcip substrate

Paraffin sections were mounted on Super frost + glass slides and deparaffinized. The slides were then treated with proteinase-K 10 μg/ml at 37 °C for 10 min, pre-hybridizd in Exiqon hybridization buffer (Exiqon, Vedbæk, Denmark) at 37 °C for 2 h, hybridized with 50 nM miRNA-30c probe and washed stringently with 5 × SSC, 1 × SSC and 0.2 × SSC buffers at 37 °C for 30 min. DIG blocking reagent (Roche, Mannheim, Germany) was added for 1 h at 37 °C in maleic acid buffer with 2% sheep serum. After which, alkaline phosphatase-conjugated anti-digoxigenin was added at 4 °C for 12 h (1:500 in Roche blocking reagent). 4-nitroblue tetrazolium (NBT) and 5-brom-4-chloro-3′-Indolylphosphate (BCIP) substrate (Roche) were used for enzymatic development to form dark-blue NBT-formazan precipitates at 37 °C for 60 min. The sections were lightly counterstained with nuclear fast red (Vector Laboratories, Burlingname, CA) at 25 °C for 1 min and mounted. The expression of miR-30c was detected, using digoxin-labelled locked nucleic acid-modified RNA probes against the full length mature miR-30c sequence. MiR-30c probe sequences are listed in Table S1.

The miRNA-126 ISH was performed essentially as previously described [29 (link),30 (link)]. In brief, 6-μm thick tissue sections were pre-treated with a proteinase-K solution followed by hybridization with a double 6-carboxyfluorescein (FAM)-labelled Locked Nucleic Acid (LNA) miRCURY probe (LNA™ microRNA detection probe, Exiqon A/S, Denmark). After stringent washes in SSC buffers, the probes were detected with alkaline phosphatise-conjugated sheep anti-FAM Fab fragments (Roche). The 4-nitro-blue tetrazolium (NBT) and 5-brom-4-chloro-3′-Indolyl-phosphate (BCIP) substrate (Roche) were added and samples were incubated leading to a blue precipitate (the ISH signal). Counterstaining was performed with nuclear fast red. All slides were processed in a Tecan Freedom Evo automated hybridization instrument (Tecan, Männedorf, Switzerland) [30 (link)] in series of 48 slides per run.