Dako immunofluorescence mounting medium

Mouse brains were fixed in 4% paraformaldehyde and subsequently paraffin-embedded by standard protocols. AT sections (7 µm thick) were deparaffinized in xylene and rehydrated through descending grades of ethanol to water. Antigen retrieval was performed in Tris/EDTA buffer at pH 9.5 twice for 5 minutes at 95 C. Sections were rinsed in PBS supplemented with 0.3% Triton-X (Sigma-Aldrich, St Lois, Missouri, USA) 3 times for 5 minutes. Unspecific binding sites were blocked using 1% bovine serum albumin in PBS supplemented with 0.3% Triton-X for 30 minutes at room temperature. The primary antibodies chicken anti-GFP (1:100; Abcam) and rabbit anti-Iba1 (1:200; WAKO; Richmond, VA) were incubated overnight at 4°C. To detect the primary antibodies, fluorochrome-conjugated secondary antibodies (1:200 each; all from Invitrogen; Karlsruhe, Germany) were applied for 1 h at RT. Autofluorescence of the tissue was quenched by using pre-warmed 0.3% sudan black for 2 minutes. Nuclear counterstain was performed by using 4,6-diamidin-2-phenylindol (DAPI; 1:10,000 in PBS) for 5 minutes, followed by 3 buffer rinses. Finally, sections were embedded with Dako immunofluorescence mounting medium (DAKO, Glostrup, Denmark). Images were taken using a FV1000 confocal laser scanning microscope (Olympus, Hamburg, Germany).