Immunofluorescent double labelling for S100β and Fluorescent In Situ Hybridization (FISH) for C3 on 10 μm thick frozen sections of human tissue fixed with 4% PFA and cryopreserved in 30% sucrose (see tissue sample details in Supplemental Data Tables 4, 6, 7). To generate the C3 antisense RNA probe C3 was synthesized from the pCMV SPORT6 C3 plasmid (Open Biosystems reference MHS6278-202800305), digested with SalI and RNA was transcribed from the T7 promoter. Dioxigenin labeling was carried out using an RNA labeling kit (Roche) before performing alkaline hydrolysis at 60°C for 16 min. A sense probe was also generated from the same plasmid but in this case digestion was carried out with Xhol and RNA transcribed form the SP6 promoter. For ISH tissue sections were incubated with RNA probes overnight at 64 °C, and then detected with anti-Digoxigenin antibodies (Roche). Staining was amplified using a TSA staining Kit (Perkin Elmer). Immunostaining for S100β (1:1000 DAKO Z0311) was subsequently performed as described above for IHC with GFAP and C3.
