In Situ Hybridization of NBPF Probes

In situ hybridization was performed using a Tecan in situ hybridization instrument (Tecan, Männedorf, Switzerland) essentially as described elsewhere ^{[10 (link)]} . In brief, 6-μm thick tissue sections were pre-digested with proteinase-K (25μg/ml for 8 minutes at 37°C). In situ hybridization was performed by incubating the two double-FAM labeled NBPF LNA probes mixed or separately at 40 or 60 nM diluted in Exiqon hybridization buffer at 57°C for 1 hour. Double-FAM labeled scramble (60 nM) and miR-126 (at 60 nM) probes were used as negative and positive controls, respectively. After stringent washes in SSC buffers, the sections were incubated with alkaline phosphatase – conjugated anti-FAM (1:800, Roche, Mannheim, Germany). Slides were developed in 4-nitro-blue tetrazolium (NBT) and 5-brom-4-chloro-3’-Indolyl-phosphate (BCIP) substrate (Roche) for 60 minutes resulting in a dark-blue precipitate. Slides were counter stained with nuclear fast red (Vector Laboratories, Burlingname, CA) if not otherwise stated.