Additional hippocampal tissue from the same patients and from four non-HS cases was fixed in 10% buffered formalin and embedded in paraffin. Paraffin-embedded tissue was sectioned at 6 μm, mounted on pre-coated glass slides (Star Frost, Waldemar Knittel GmbH, CH5424802 Brunschweig, Germany) and organosilane-coated slides (SIGMA, St Louis, MO, USA), and two slices were used for in situ hybridizations and immunocytochemistry. Two additional slices were used

for the double-staining, combining in situ hybridization with immunocytochemistry (in the same slices) with different antibodies, as described below. Additional immunocytochemistry (single-labelling) was performed for complement factor H (CFH) in both control and HS

hippocampal tissue. For RNA isolation, frozen material was homogenized in Trizol LS Reagent (Invitrogen, Carlsbad, CA, USA). After addition of 200 μg glycogen and 200 μL chloroform, the aqueous phase was isolated using Phase Lock tubes (Eppendorf, Hamburg, Germany). RNA was precipitated with isopropyl alcohol, washed with 75% ethanol and dissolved in water. The concentration and purity of RNA were determined at 260/280 nm using a nanodrop spectrophotometer (Ocean Optics, Dunedin, FL, USA). cDNA was generated using Taqman MicroRNA reverse transcription kit (Applied Biosystems, Foster City, CA, USA) according to manufacturer’s instructions. miRNA (miR-146a and the U6B small nuclear RNA gene, rnu6b) expression was analysed using Taqman microRNA assays (Applied Biosystems), which were

run on a Roche Lightcycler 480 (Roche selleck chemical Applied Science, Basel, Switzerland) according to the instructions of the manufacturer. Data analysis was performed with the software provided by the manufacturer. Statistical analyses were performed with spss for Windows (spss 11.5, SPSS, Chicago, IL, USA) using two-tailed Student’s t-test, and to assess differences between more than two groups a non-parametric Janus kinase (JAK) Kruskal–Wallis test followed by Mann–Whitney U-test were used. A value of P < 0.05 was considered significant. In situ hybridization for miR-146a was performed using a 5′ fluorescein-labelled 19mer antisense oligonucleotide containing locked nucleic acid and 2′OME RNA moieties (FAM – AacCcaTggAauTcaGuuCucA, capitals indicate LNA, lower case indicates 2′OME RNA). The oligonucleotides were synthesized by Ribotask ApS, Odense, Denmark. The hybridizations were done on 6-μm sections of paraffin-embedded materials described previously (Budde et al., 2008). The hybridization signal was detected using a rabbit polyclonal anti-fluorescein/Oregon green antibody (A21253, Molecular Probes, Invitrogen) and a horseradish peroxidase-labelled goat anti-rabbit polyclonal antibody (P0448 Dako, Glostrup Denmark) as secondary antibody.