In Situ Hybridization of miR-21 in MF Skin

Six μm thick tissue sections from paraffin embedded lesional MF skin biopsies were used for in situ hybridization as described elsewhere [50 (link), 73 (link)]. In brief, the slides were deparaffinized and placed in a Tecan Freedom Evo automated hybridization instrument (Tecan, Männedorf, Switzerland), which was programmed to perform the following steps: proteinase-K treatment using 15 μg/mL for 8 min at 37°C, pre-hybridization in formamide-free hybridization buffer (Exiqon, Vedbæk, Denmark) at 57°C for 15 min, in situ hybridization with double-FAM-labeled miR-21 and scramble LNA probes (both at 40nM, Exiqon) at 57°C for 60 min, stringent washes with SSC buffers at 57°C over 33 min followed by incubation of alkaline phosphatase-conjugated anti-FAM, NBT-BCIP substrate (all from Roche, Mannheim, Germany), and finally nuclear fast red counterstain (Vector Laboratories, Burlingname, CA, USA). Finally, all slides were dehydrated and the sections mounted with Eukitt (Electron Microscopy Sciences, Hatfield, PA, USA).