Mice were euthanized by intra-cardiac perfusion and brains were fixed in neutral buffered formalin. Paraffin-embedded brain sections were assessed with the following antibodies: GFAP (Cell Signaling, 1:1000), Iba1 (Wako, 1:1000); pSer129/81A αSyn (1:3000; [13 (link), 36 (link)]); EP1536Y (AbCam, 1:1000); p62 (Protein Tech; 1:1000), cd11b (AbCam; 1:250), MHCII (Novus; 1:50) and NeuN (Abcam; 1:500). For all antibodies except cd11b and MHCII, antigen retrieval was performed by steaming for 25 min in water. For cd11b and MHCII, antigen retrieval was done by steaming in Dako Target Retrieval Solution S1699 (modified citrate buffer, pH 6.1). Colorimetric slides were treated with ImmPress reagents (Vector Labs) and visualized with 3, 3’diaminobenzidine followed by hematoxylin counterstaining. Fluorimetric slides were visualized with Alexa Fluor conjugated secondary antibodies (Invitrogen) and counterstaining with 4’, 6-diamidino-2-phenylindole (DAPI; Southern Biotech). All colorimetric slides were scanned using the Aperio XT whole slide scanner while fluorescent slides were visualized using Olympus BX60 microscope with a color digital camera.
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