Dm6000b

Double-immunofluorescence stainings were performed on a TMA containing tissue from nine GBMs, normal colon, placenta, cerebellum, and rat hippocampus as previously described [20 (link), 28 (link), 29 (link)]. The preparations as well as the first step in the stainings were as described above. After detection of TfR1 (CD71) (1+200), FTH (1+12000), or FTL (1+16000) using Catalyzed Signal Amplification II kit with FITC (CSA II, Dako), a second round of HIER followed by quenching of endogen peroxidase was performed. Sections were then incubated with antibodies against nestin (96908, 1+200, R&D systems, Minneapolis, Minnesota, USA), CD133 (W6B3C1, 1+40, Miltenyi Biotec, Teterow, Germany), GFAP (Z0334, 1+8000, Dako), or IBA-1 (019–19741 1+12000, Wako Pure Chemical Industries, Osaka, Japan). Tyramide Amplification Signal Cyanine 5 (TSA-Cy5, Perkin Elmer, Waltham, Massachusetts, USA) was used as detection system. Sections were mounted with VECTASHIELD Antifade Mounting Medium with 4.6-diamidino-2-phenylindole (DAPI, VWR International, Radnor, Pennsylvania, USA). Fluorescent images were taken with a Leica DM6000B microscope connected to an Olympus DP72 1.4 Mega Pixel CCD (Olympus, Tokyo, Japan) camera using DAPI (Omega XF06, Omega Optical, Brattleboro, Vermont, USA), FITC (Leica, Wetzlar, Germany) and Cy5 (Omega XF110-2) filters.