Vectamount medium

Immunohistochemistry was performed according to the protocol previously described [15 ] with slight modification. Both paraffin and cryo-sections were used for immunohistochemistry and immunofluorescence with similar anatomic features (see the “Supplemental Methods” for details). The primary antibodies were anti-myelin basic protein (MBP) (C-16 clone, SC-13914, Santa Cruz, CA, USA; 1:100 dilution); rat anti-platelet endothelial cell adhesion molecule [PECAME-1 (CD31), BD no. 550274, USA,1:200 dilution], with mouse monoclonal anti-α-smooth muscle actin [α-SMA, SC-53142, Santa Cruz, CA, USA; 1:50 dilution]; and anti-platelet-derived growth factor [PDGFR-β (958): SC-432, Santa Cruz, CA, USA; 1:200 dilution], isolectin B4 conjugates (Invitrogen, molecular probe Life Technology, IB4 no. 121411). For biotinylated immunostaining, the brain sections were incubated in the anti-MBP primary antibodies and using the avidin–biotin–peroxidase complex method with diaminobenzidine (DAB) as the chromogen. The immunostaining was carried out using the ABC kit system (Vector, Burlingame, CA, USA). After staining, the sections were counterstained with Harris hematoxylin (cat. no. HHS35-1L; Sigma) for few seconds. The sections were then dehydrated rapidly through ethanol and xylene and mounted with VectaMount medium (Vector).

Differentiated control or APPswe-expressing SH-SY5Y cells and primary neurons were fixed with 4% paraformaldehyde (PFA) for 10 min, washed twice with 1X PBS, and then permeabilized with 0.5% (cells) or 0.3% (neurons) Triton X-100 for 5 min. Non-specific binding sites were blocked for 1–2 h in 3% PBS-BSA containing 0.05% tween at room temperature. Primary antibodies were diluted (Supplementary Table 1) in PBS-BSA 0.3% containing 0.005% tween and applied overnight (ON). After three washes with 1xPBS, AlexaFluor-conjugated secondary antibodies (Jackson ImmunoResearch) were applied for 1 h. Nuclei were stained with DAPI (1/10000, Invitrogen) and slides were mounted with Vectamount medium (Vector). Images were taken with a confocal Leica TCS SP5 microscope or LSM 780. The colocalization quantification was determined using the Fiji plug-in JACoP (Just Another Colocalization Plug-in).

The left lung lobe was inflated with 10% formalin (Fisher SF98-4), fixed overnight, and kept in 70% ethanol until processing. Paraffin-embedded tissue was sectioned (5 µm) and stained using hematoxylin and eosin (H&E) or IHC antibodies and scored in a blinded fashion by an experimental pathologist. IHC was performed using primary antibodies against Ly6G (1:250, BD Biosciences 551459) and S. pneumoniae (1:1000; Novus Biologicals NB100-64502), and detected using ImmPress polymer reagents (Vector Laboratories). Antigen expression was visualized with 3,3’Diaminobenzidine and alkaline phosphatase according to manufacturer’s protocol (Vector Laboratories). Sections were counterstained with Hematoxylin QS and slides were coverslipped using VectaMount medium (Vector Laboratories).