Ab109497

The remaining hemispheres from NBH (controls), ME7 and ME7 + JNJ527 mice (n = 7–8/group) were cut at 35 µm thickness using the HM 430 Sliding Microtome (Thermo Fisher Scientific).
Immunohistochemistry for TSPO (ab109497, Abcam) and triple immunofluorescence of TSPO (ab109497, Abcam), Iba1 a marker for both microglia and macrophages (ab5076, Abcam) and GFAP astrocytes (ab4674, Abcam); along with triple immunofluorescence of TSPO (ab109497, Abcam), NeuN for neurons (266006, Synaptic Systems) and CD31 for vascular endothelium (AF3628, R&D Systems) were performed to determine brain TSPO protein expression and TSPO cellular sources.
Free floating sections were incubated in citrate buffer pH = 8 at 80 °C for 30 min and 0.3% v/v H2O2 to block for endogenous peroxidase activity (only for bright field immunohistochemistry), and with 10% w/v milk and 0.3% v/v Triton X‐100 in TBS for 40 min. After rinses with tris buffered saline (TBS), sections were incubated overnight at 4 °C with rabbit anti‐TSPO antibody (1:10,000 in blocking solution, ab109497, Abcam) for bright field immunohistochemistry, and with a mix of primary antibodies for immunofluorescence (see Additional file 1: Table S1). After washes with TBS, sections were incubated with the appropriate biotinylated (Vector Labs) or Alexa-conjugated secondary antibodies (see Additional file 1: Table S1).

5 μm paraffin sections were cut from mouse brains fixated in 4% paraformaldehyde and subsequently immunohistochemically stained and analysed.
Primary antibodies and dilutions used: anti-PBR antibody (anti-PBR, TSPO) (rabbit, 1:250, ab109497, Abcam, Cambridge, UK), anti-ionized calcium binding adapter molecule 1 (anti-Iba-1) (rabbit, 1:500, 019-19741, Wako Chemicals USA, Inc. Richmond, VA, USA), anti-glial fibrillary acidic protein (anti-GFAP) (chicken, 1:500, ab4675, Abcam, Cambridge, UK), anti-F4/80 (rat, 1:200, ab6640, Abcam, Cambridge, UK) and CD163 (rabbit, 1:50, bs-2527R, Bioss). Tissue sections were investigated with a combined fluorescent-light microscope (Nikon Eclipse NI-E, Nikon, Tokyo, Japan). Details are given in Supplementary Material and Methods.

TSPO protein levels of were analyzed under non-reducing conditions. For this, tissue samples and primary microglia were lysed by sonication in ice cold PBS supplemented with protease and phosphatase inhibitors (Complete protease inhibitor cocktail, Roche). Protein concentration was determined by BCA Protein Assay according to the manufacturer’s instructions (Thermo Scientific). Equal amounts of samples were heated in SDS sample buffer containing only 0.2% SDS and no β-mercaptoethanol and then loaded onto 12% tris–glycine polyacrylamide gels and run under standard conditions. For immunoblotting, proteins were electrophoretically transferred onto a 0.45 µm nitrocellulose membrane (Bio Rad) at 100 V for 1 h. Membranes were blocked with 5% nonfat dried milk powder in TBS‐T before incubation with primary antibody against TSPO (1:1000 dilution, rabbit polyclonal, ab109497, Abcam) or Actin (1:1000 dilution, chicken anti‐b‐Actin clone AC‐15, A5441, Sigma‐Aldrich). After several washing steps in TBS-T, membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies (1:4000 dilution, Dako) and the immune complex was visualized by a MultiImage II system (Alpha Innotech). PageRuler pre-stained protein ladder (Thermo Scientific) was used for identification of protein size. Band intensities were quantified using ImageJ.