Foetal calf serum

Murine peritoneal cavity cells (PerC) were obtained by peritoneal lavage. Peritoneal lavage was performed by intraperitoneal injection of 10 mL ice-cold PBS supplemented with either 2% foetal calf serum (Biochrom, Berlin, Germany) for RNA isolation or 0.5% fatty-acid-free bovine serum albumin (BSA, Sigma-Aldrich, St. Louis, MO, USA) for cell culture experiments involving later use of spinghosine-1-phosphate (S1P). Absolute cell numbers were determined using BD Trucount tubes (BD Biosciences, Becton Dickinson, Franklin Lakes, NJ, USA).

Double immunofluorescence stainings were carried out manually on selected tumour specimens using antibodies against Sox9 and β-catenin, Olig2 or GFAP (monoclonal mouse anti GFAP; 1:500; clone 6F2; Dako; Glostrup, Denmark) as well as Sox2 and Olig2 or GFAP. After dewaxing the slides, antigen retrieval by microwave pre-treatment was performed in citrate-buffer at pH 6. To prevent unspecific antibody binding, the slides then were incubated for two hours with a blocking solution containing PBS with foetal calf serum (5%; Biochrom AG; Berlin, Germany), goat serum (3%; Millipore; Temecula, CA, USA) and Triton ×100 (0,1%; Sigma-Aldrich; Steinheim, Germany). Primary antibodies were diluted in the blocking solution and incubated overnight at 4 °C. Carbocyanine 2 (Cy2) goat anti-mouse or goat anti-rabbit (1:100; green; Dianova; Hamburg, Germany) and Cy-3 goat anti-rabbit or goat anti-mouse secondary antibodies (1:100; red; Dianova; Hamburg, Germany) served as fluorescent markers, while cell nuclei were counterstained with Hoechst 33342 (Sigma Aldrich; Steinheim, Germany) at a concentration of 500 ng/ml for 5 min at room temperature. Slides were analysed using an Olympus BX-51 fluorescent microscope (Olympus; Hamburg, Germany) equipped with an F-View II CCD camera (Soft imaging systems; Stuttgart, Germany).

Species-identified and pooled mosquitoes, with up to 50 individuals per pool, were transferred into Lysing Matrix D tubes (Peqlab, Erlangen, Germany) containing 1 ml of M199 tissue culture medium, antibiotics, antimycotics solution (ABAM, Invitrogen), and 2% foetal calf serum. The mosquito-fluid mixture was homogenised by two rounds of reciprocation at 5 m/sec for 15 seconds with cooling on ice water between the rounds (FastPrep FP120, BIO 101, Q-BIOgene). The homogenate was pressed through a 45 μm filter (Millipore), before additional 1 mL M199 was pressed through the filter, and 0.2 mL of the filtrate was inoculated into one well of a 6-well plate with confluent Vero B4 cells (LGC Standard, Wesel, Germany). Cells were provided 3 mL M199 with 2% foetal calf serum (Biochrom AG, Berlin, Germany) and incubated at 37°C in 5% CO₂. Cell cultures were inspected daily, for at least one week, and from wells with observed cytopathogenic effects 0.5 mL of culture supernatant was inoculated into a 25 cm² flask with Vero cells for confirmation of virus-induced cytopathogenic activity.