Brain slices (5-10 µm) were post fixed in 4% PFA and processed for immunohistochemistry for the detection of the translocator binding protein (TSPO), myelin basic protein (MBP), astrocytes using glial fibrillary protein (GFAP), and microglia/ macrophages using ionized calcium-binding adapter molecule 1 (Iba-1) markers, as previously described 10 (link), 26 (link). Immunofluorescence or immunoperoxidase staining was performed in one section of at least four randomized animals using the following primary and secondary antibodies:
TSPO (1:250, rabbit anti-TSPO, NBP1-95674, AB_11015478, Novus Biologicals, Cambridge, UK), myelin basic protein (MBP) (1:200, chicken anti MBP, AB9348, RRID:AB_2140366, Merck, Darmstadt, Germany) , Iba1 (1:250, rabbit anti α Iba1, 019-19742, RRID:AB_2314666; Wako Chemicals, Neuss, Germany), GFAP (1:1000, chicken anti GFAP, ab4674, RRID:AB_304558, abcam, Cambridge, UK); Alexa Fluor 488/555 (1:800; Life Technologies), DSB-X™ Biotin Goat Anti-Chicken IgG (1:800; Life Technologies). Double immunofluorescence for TSPO and Iba-1 was performed using a preconjugated TSPO antibody (1:100; Anti-PBR antibody [EPR5384] (Alexa Fluor® 647) (ab199836)). Slices incubated with only secondary antibodies served as negative controls. Images were acquired with a combined fluorescence- light microscope (Nikon Eclipse NI-E, Nikon, Tokyo, Japan).
TSPO (1:250, rabbit anti-TSPO, NBP1-95674, AB_11015478, Novus Biologicals, Cambridge, UK), myelin basic protein (MBP) (1:200, chicken anti MBP, AB9348, RRID:AB_2140366, Merck, Darmstadt, Germany) , Iba1 (1:250, rabbit anti α Iba1, 019-19742, RRID:AB_2314666; Wako Chemicals, Neuss, Germany), GFAP (1:1000, chicken anti GFAP, ab4674, RRID:AB_304558, abcam, Cambridge, UK); Alexa Fluor 488/555 (1:800; Life Technologies), DSB-X™ Biotin Goat Anti-Chicken IgG (1:800; Life Technologies). Double immunofluorescence for TSPO and Iba-1 was performed using a preconjugated TSPO antibody (1:100; Anti-PBR antibody [EPR5384] (Alexa Fluor® 647) (ab199836)). Slices incubated with only secondary antibodies served as negative controls. Images were acquired with a combined fluorescence- light microscope (Nikon Eclipse NI-E, Nikon, Tokyo, Japan).