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For 18E6 analysis, which recognizes a unique epitope within the immunoglobulin (Ig)–like domain of Arg-gingipain (RgpB) (58), IHC was performed using the Ventana Benchmark XT automated slide preparation system at the UCSF Brain Tumor Research Center tissue core. For immunoperoxidase staining, after tissue sections (5 μm thickness) were deparaffinized (at 75°C; EZ-Prep, Ventana Medical Systems), antigen retrieval was performed for 30 min [Cell Conditioning 1 (pH 8.5), Ventana Medical Systems] at 95° to 100°C. H2O2 (3%) (Thermo Fisher Scientific) was applied for 8 min to reduce background staining. Antibody 18E6 (University of Georgia Monoclonal Antibody Facility) was incubated at RT for 32 min at 1:10 dilution. Staining was developed using the UltraView Universal DAB Detection System (Ventana Medical Systems), and slides were counterstained with hematoxylin.

For immunofluorescence, sections were deparaffinized in xylene and rehydrated in a graded alcohol series. Heat-mediated antigen retrieval was performed with citric buffer (pH 6.0) (H-3300, Vector Laboratories). After PBS washes, sections were incubated in blocking solution, 5% donkey serum, and 0.3% Triton X-100 in PBS for 1 hour at RT. Sections were then incubated overnight at 4°C in primary antibodies anti-MAP2 (1:500; ab5392, Abcam), CAB101 (1:500), anti-IBA1 (1:1000; ab97120, Abcam), anti–β-amyloid, 17–24 (1:500; SIG-39200; 4G8, BioLegend), and anti-AT8 (1:2000; MN1020B, Thermo Fisher Scientific) in 3% donkey serum and 0.3% Triton X-100 in PBS. After PBS washes, slides were incubated with secondary antibody solution, either Alexa Fluor 647 goat anti-chicken (1:200; A21449, Life Technologies) and Alexa Fluor 488 donkey anti-rabbit (1:200; Jackson ImmunoResearch) mixed with anti–GFAP-Cy3 (1:250; MAB3402C3, Millipore) or Cy3-donkey anti-rabbit (1:200; Jackson ImmunoResearch), Alexa Fluor 488 donkey anti-mouse (1:200; Jackson ImmunoResearch), and Alexa Fluor 647 donkey anti-goat (1:200; Jackson ImmunoResearch) in PBS with 0.3% Triton X-100 for 2 hours at RT. Sections were washed in PBS and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen; D1306). Autofluorescence was quenched with TrueBlack Lipofuscin Autofluorescence Quencher 1:20 in 70% ethanol (catalog no. 23007, Biotium), and slides were mounted with ProLong Gold Antifade (P36930, Thermo Fisher Scientific). Coimmunofluorescence on P. gingivalis was performed by drying bacteria on SuperFrost Plus microscope slides. Bacteria were immersed in 4% paraformaldehyde for 10 min, washed three times in PBS, and incubated in formic acid for 7 min, followed by another three washes with PBS. Cells were exposed to anti–β-amyloid, 17–24 (1:500; SIG-39200; 4G8, BioLegend) and CAB101 (1:500) in 3% donkey serum and 0.3% Triton X-100 in PBS for 30 min at RT, followed by 30-min incubation in Cy3-donkey anti-rabbit (1:200; Jackson ImmunoResearch) and Alexa Fluor 488 donkey anti-mouse (1:200; Jackson ImmunoResearch) in PBS, counterstained with DAPI, and coverslipped with ProLong Gold Antifade (Thermo Fisher Scientific; P36930).

Histological analysis was performed on an Olympus BX61 motorized microscope. Fluorescence images were taken with a sCMOS camera (Zyla-5.5-USB3, Andor), and brightfield images were taken on a color charge-coupled device camera (DP27, Olympus). Images were processed for brightness and contrast correction, cropping, and addition of scale bars with CellSens 1.14 Dimension software (Olympus).

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