Immunohistochemistry and immunofluorescence

After the last imaging examination, mice were sacrificed and perfused with 0.9% NaCl and 4% PFA. Brains were fixed in 4% PFA O/N, embedded in paraffin and cut in 10 µm coronal sections. Immunohistochemistry was performed for all animals using the paraffin embedded coronal brain sections employing antibodies for microglia (1:250, rabbit anti Iba1, 019-19741, Wako), TSPO (1:250, rabbit anti PBR, ab109497, Abcam, Cambridge UK) and GFAP (1:500, chicken anti GFAP, ab13970, Abcam, Cambridge UK). Antigen retrieval was performed by boiling the slides in citrate buffer (pH 6; 18 min). Slides were then treated with blocking solution at RT for one hour (1% BSA and 0.5% Triton-X in PBS), subsequently incubated (4 °C O/N) with the primary antibodies, followed by incubation with Alexa Fluor 488-conjugated anti-rabbit secondary antibody (1:1000, A-21206, life technologies, Carlsbad, USA), or Alexa Fluor 555-conjugated anti-rabbit (1:1000, A-21432, life technologies, Carlsbad, USA). Nuclei were stained with DAPI (0.2 µg/ml in PBS, Roth, Karlsruhe, Germany). Slices were embedded using Mowiol solution (6 g glycerol, 2.4 g Mowiol 4-88 (0713, Roth, Karlsruhe, Germany), 6 ml distilled water, 12 ml Tris-HCl (pH8.5)). For conventional histology, slides were incubated with a biotinylated goat anti-rabbit (1:800, 45 min, {"type":"entrez-nucleotide","attrs":{"text":"B21078","term_id":"2396132","term_text":"B21078"}}B21078, Life Technologies, Darmstadt, Germany), followed by HRP-Streptavidin incubation (1:600, 20 min, K1016, Dako, Hamburg, Germany). The staining was visualized by incubation with 3,3 Diaminobenzidine (D-5637, Sigma, St. Louis, USA) for 5 min. Sections were counterstained with hematoxylin, dehydrated and mounted using Entellan (Merck, Darmstadt, Germany). Images were acquired with a combined fluorescence-light microscope (Nikon Eclipse NI-E, Nikon, Japan). Positive cells were quantified by manually counting the number of cells present in 20× magnification images taken from biological triplicates.