*4.2. Brain Tissue Preservation and Fixation*

All brains were processed at autopsy in a standardized manner [52]. The median postmortem interval for autopsies in the BBDP was 3.8 h. After brain removal, the cerebellum and brain stem are separated from the hemispheres, then each brain is sectioned in a frame into 1cm thick coronal slabs. The hemispheres are divided, with the left hemisphere being frozen on dry ice for storage at −80 ◦C, and the right hemisphere being fixed for 48 h in buffered formalin solution. After fixation, the coronal pieces are rinsed and transferred to a phosphate-buffered solution of 15% glycerol/15% ethylene glycol as cryoprotectant. Brain regions used for subsequent studies are dissected from frozen or fixed coronal slices by experienced neuroanatomists.

#### *4.3. Neuropathological Diagnosis Criteria*

All donated brains received full neuropathological diagnosis including reference to pre-mortem clinical history of each case. Consensus clinical and neuropathological criteria were used to diagnose AD, Dementia with Lewy bodies (DLB) and PD in these cases [53,54]. To assess severity of AD pathology in each case, tissue sections from 5 brain regions (entorhinal cortex, hippocampus, frontal cortex, temporal cortex and parietal cortex) were stained with Thioflavin-S, Gallyas or Campbell–Switzer histological stains and assessed semi-quantitatively for the density of neurofibrillary tangles and amyloid plaques with each brain region being ranked on a scale of 0–3. By combining the measures across these 5 brain regions, assessment of AD pathology was ranked on an ordinal scale of 0–15 for plaques and tangles [55]. The two sets of cases used in this study were subdivided into non-demented low plaque (LPND) (plaque score < 6), non-demented high plaque (HPND)(plaque score 6–14) and AD with dementia (plaque score > 12).

### *4.4. Peroxidase*/*Diaminobenzidine Immunohistochemistry*

Formaldehyde-fixed tissue sections (25 μm) from middle temporal gyrus (MTG) from 36 cases cut on a sliding microtome were used for cellular localization of purinergic receptor P2RY12 in relation with AD pathological markers, amyloid-beta (Aβ and phosphorylated tau, or markers of microglia activation (HLA-DR, and IBA-1). Sections of hippocampus from 4 cases (2 LP, 2 AD) were also used for comparison. A free-floating immunohistochemistry method was used. Tissue sections from a series of cases were rinsed three times with phosphate-buffered saline-Triton X100 (PBSTx) (0.1 M phosphate Buffer, pH 7.6, 0.137 M NaCl, 0.3% Triton X100) and pretreated with 1% hydrogen peroxide in PBSTx for 30 min to quench endogenous tissue peroxidases. For certain antibodies, antigen retrieval was carried out by heating sections in 10 mM EDTA (pH 8.0) at 80 ◦C for 30 min and then cooling to room temperature for 30 min. Sections were incubated free-floating at room temperature for 18 h with shaking in PBSTx with optimal dilution of antibodies. To identify localized antibodies, sections were sequentially washed three times in PBSTx for 10 min, incubated in biotinylated secondary antibody (1:1000), sequentially washed again three times in PBSTx and then incubated in preformed avidin-biotin horseradish peroxidase enzyme complex (ABC-Vector Laboratories, Burlingame, CA USA) solution (1:1000) for 1 h. Sections were then washed three times in PBSTx and two times in 50 mM Tris-HCl (pH 7.6) before incubation in peroxidase substrate. Most frequently used was nickel ammonium sulfate-enhanced diaminobenzidine as substrate to produce a purple reaction product (50 mM Tris-HCl, pH 7.6, 1% saturated nickel ammonium sulfate, 40 mM imidazole, 100 μg/mL diaminobenzidine-HCl (Dojindo, Kumamoto, Japan) and 0.0003% hydrogen peroxide). For two-color immunohistochemistry, reacted sections were rinses in PBSTx, treated with 1% hydrogen peroxide to remove residual peroxidase activity and then incubated

for a second time in primary antibody overnight at room temperature. The detection procedure followed the above described protocol except the substrate used was diaminobenzidine without nickel ammonium sulfate as substrate to produce a brown reaction product (50 mM Tris-HCl, pH 7.6, 20 mM imidazole, 200 μg/mL diaminobenzidine-HCl and 0.0006% hydrogen peroxide). Reacted sections were mounted on slides, counterstained in most cases with 0.5% neutral red, dehydrated, cleared and coverslipped using Permount mounting media (ThermoFisher, Waltham, MA, USA Sections used for quantitative measurements were not counterstained.

#### *4.5. Fluorescent Confocal Immunohistochemistry*

Multiple-color fluorescent confocal immunohistochemistry was carried out for antibody pairs to verify cellular co-localization of antigens with P2RY12-expressing cells [19]. Tissue sections were incubated with optimal dilutions of antibodies at room temperature overnight with shaking. After three washes (10 min each) in PBSTx, sections were incubated in the dark with optimal concentrations of fluorescent-labeled secondary antibodies. Bound primary antibodies were detected with Alexa Fluor 488 (donkey anti-goat IgG), Alexa Fluor 568 (donkey anti-rabbit or anti-mouse IgG) or Alexa Fluor 647 (donkey anti-mouse IgG) (ThermoFisher, San Jose, CA, USA). After washing and mounting, sections were counterstained with Sudan Black (1% solution in 70% ethanol for 10 min) to quench tissue autofluorescence, destained with 70% ethanol, and stained with DAPI to reveal nuclei. Sections were coverslipped using antifading-hardening-fluorescent mounting media (Vector Labs, Burlingame, CA, USA). Sections were imaged using an Olympus FV1000 confocal microscope, and compiled Z-scans obtained and processed using Olympus microscope system software (Olympus Corporation, Tokyo, Japan). Some sections were imaged using a Leica SP8 confocal microscope system (Leica-Microsystems, Wetzlar, Germany).

#### *4.6. Antibodies*

The following primary antibodies were used in this study: P2RY12 (Novus Biologicals, Centennial, CO, USA) catalog no. NBP2-33870; rabbit, 1:1000–1:2000 used for immunohistochemistry (IHC) and western blot (WB). P2RY12 (Alomone Labs, Tel Aviv, Israel); catalog no. APR-012; rabbit, 1:200 used for IHC. P2RY12 (Abcam, Cambridge, UK); catalog no. AB83066; rabbit, 1:2000 used for WB. HLA-DR clone LN3 (Abcam); catalog no. AB80658; mouse, 1:750 used for IHC. CD68 (Biolegend, San Diego, CA, USA); catalog no 916104; mouse, used at 1:250 for IHC. Progranulin (R&D Systems, Minneapolis, MN, USA; catalog no. AF2420, goat used at 1:100 for IHC. IBA-1 (Wako, Richmond, VA, USA); catalog no. 019-19741; rabbit, 1:1000 used for IHC. Aβ clone 6E10 (Biolegend); catalog no. 803001; mouse, 1:2000 used for IHC. pTau clone AT8 (ThermoFisher, Waltham, MA, USA); catalog no. MN1020: mouse, 1:3000 used for IHC.

Secondary biotinylated-antibodies used for enzyme histochemistry and Avidin-Biotin-Complex (ABC) peroxidase were obtained from Vector Labs (Burlingame, CA, USA). Fluorescent-labeled secondary antibodies used for confocal microscopy, and horseradish peroxidase (HRP)-conjugated secondary antibodies used for western blots were obtained from ThermoFisher (Waltham, MA, USA).

#### *4.7. Verification of Antibody Specificity*

The majority of the immunohistochemistry studies reported in this communication were carried out with the Novus P2RY12 antibody. Peptide absorption studies were carried out using a recombinant 40-amino acid protein (NBP2-33870PEP, Novus), the immunizing peptide for the Novus antibody (NBP2-33870). P2YR12 antibody (1:1000) was mixed with 20-fold molar excess of protein for 18 h, and these materials were used to stain sections from three separate cases in parallel with non-absorbed antibody. In addition, comparisons of immunostaining patterns were carried using an independent antibody to P2RY12 (1:250, Alomone Labs), prepared against an 18-amino acid peptide sequence that did not overlap with the immunizing sequence of the Novus antibody.

#### *4.8. Brain Sample Extraction and Western Blot*

Brain tissues samples (middle temporal gyrus – MTG) were dissected frozen and then further trimmed so samples being analyzed primarily contained gray matter. Detergent-soluble extracts were prepared by gently sonicating each tissue sample in 5 volumes (weight to volume) of RIPA buffer (20 mM Tris-HCl, pH 7.5. 150 mM NaCl, 1% Triton X100, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate) supplemented with protease and phosphatase inhibitors (Nacalai-Tesque, Kyoto, Japan). After 30 min incubation on ice, samples were centrifuged at 15,000× *g* for 30 min. The supernatants were transferred to new tubes and total protein concentration of each extracted sample was determined using a Micro BCA assay kit (ThermoFisher, Rockford, IL, USA) with bovine serum albumin as standard. P2RY12 polypeptides were detected in MTG samples by western blots using the Novus P2RY12 antibody (1:1000). Protein samples were dissolved in SDS-sample buffer, adjusted to contain 1 μg/μL protein, denatured by heating to 90 ◦C for 10 min, centrifuged at 15,000× *g* for 10 min to remove insoluble material and then separated on 4–20% Tris-glycine precast gradient polyacrylamide gels (Nacalai-Tesque, Kyoto, Japan). Separated polypeptides were transferred to PVDF membranes (Millipore-EMD) using a semi-dry transfer apparatus. Membranes were blocked in 5% skimmed milk solution diluted in Tris-buffered saline with 0.1% Tween 20 (TBST), and incubated for 18 h in optimal dilutions of antibody solution in 2% milk in TBST. Membranes were washed in TBST and then reacted with HRP-labeled secondary antibody for 2 h. Membranes were reacted with Chemi-Lumi-One Super chemiluminescent substrate (Nacalai-Tesque, Kyoto, Japan) and sequential images captured using an ImageQuant LAS 4000 system (GE LifeSciences, Pittsburgh, PA, USA). Band intensities were measured using Image Studio Lite (LI-COR, Lincoln, NE, USA). All membranes were subsequently reacted with HRP-labeled antibody to β-actin (FujiFilm Wako Pure Chemicals, Osaka, Japan) at 1:15,000 for 1 h and imaged in the above-described manner. Band intensities were normalized for levels of β-actin in samples.

#### *4.9. Area of P2RY12 Immunoreactivity*

To measure area of P2RY12 immunoreactivity, a complete series of cases (see Table 1) were single-stained with P2RY12 antibody (Novus) using nickel-enhanced DAB to reveal dark purple reaction product. After mounting and coverslipping of sections, images at 4x magnification were captured from each section, taking four separate, random fields of gray matter for each section. Captured images were analyzed using ImageJ analysis software (NIH, Bethesda, MD, USA) (https://imageJ.net/ImageJ, version 1.52a, accessed on 5 January, 2018) to measure area of immunoreactivity occupied by reaction product on each slide. Images were converted to gray scale and thresholds adjusted to identify positively stained microglia. Mean values of area occupied between the four separate fields measured were calculated for each case, and then mean data were compiled into respective disease groups for further statistical analysis.

#### *4.10. P2RY12 Immunoreactive Cell Counts*

Further analyses were carried out to estimate numbers of P2RY12 immunoreactive microglia in each section. Using a 25-grid-square microscope eyepiece reticule (Nikon) and 20× objective, the numbers of microglia were counted in five consecutive areas through the cortex. Each field counted corresponded to 2 mm2. These areas corresponded approximately to Layer I and II, Layer III, Layer IV, Layer V, and Layer VI. For each section, three separate areas were counted where distinct cortical layers could be detected. The patterns of cortical layers was confirmed by reference to standard text. These measures allowed sampling through all cortical layers though the AD cases had thinner cortical layers. Counting criteria required the presence of a microglial cell body to be present within the area of the reticule grid. For each case, the mean total numbers of cells were calculated, and then mean and standard error of mean for all samples from each disease group.

#### *4.11. Quantitative Reverse Transcription Polymerase Chain Reaction (qPCR) Analysis of mRNA Expression.*

RNA was prepared from human brain tissue samples and human microglia using RNAeasy Plus-Mini kits (Qiagen, Valencia, CA, USA) according to the manufacturer's protocol. RNA concentrations and purities were measured using a Nanodrop 1000 spectrophotometer and RNA integrity with an Agilent Bioanalyzer and RNA 6000 Nano kits (Agilent, Santa Clara, CA, USA). Samples used for qPCR had RIN values greater than 7.0. RNA from brain samples (0.5 μg) and cultured cell samples (0.2 μg) were reverse transcribed using the Quantitect reverse transcription kit (Qiagen) with genomic DNA elimination reagent according to manufacturer's protocol. Appropriate numbers of no reverse transcriptase controls were prepared in parallel for each batch of samples. For qPCR, cDNA samples were amplified using Perfecta Fast Mix 2x reaction mixture (Quanta Biosciences, Gaithersburg, MD, USA) supplemented with 1.25 μM of Eva Green. The primers used were as follows: P2RY12 sense: AGTCCCCAGGAAAAAGGTG; P2RY12 antisense: GTTTGGCTCAGGGTGTAAGG (reference sequence NM\_022788.40). Expression results were normalized with relative levels of β-actin mRNA using primer sequences: β-actin sense: TCCTATGTGGGCGACGAG. β-actin antisense: ATGGCTGGGGTGTTGAAG. QPCR was carried out using a Stratagene Mx3000p machine and abundance of gene expression quantified relative to a standard curve of pooled samples. QPCR analyses followed recommended criteria for minimum information for publication of quantitative real-time PCR experiment (MIQE) [56].

## *4.12. Human Brain-Derived Microglia Isolation and Stimulation*

Human brain microglia were prepared from frontal cortex from three different donor cases for this study following our published procedures [9,13]. After 10–14 days in culture, microglia were replated into wells at 10<sup>5</sup> cells/well in 12-well plates prior to stimulation. For these experiments, microglia were unstimulated or treated with interleukin (IL)-4 (20 ng/mL), aggregated Aβ42 (2 μM and 5 μM), interferon-γ (IFNγ) (20 ng/mL), lipopolysaccharide (LPS) (100 ng/mL), LPS and IFNγ combined, and IL-6 (20 ng/mL) [9]. After 24 h treatment, RNA was isolated from microglia using the above described methodology. Expression of P2RY12 mRNA in treated and untreated cells were carried out as described above for brain samples. Western blot analysis for P2RY12 protein expression by IL-4 stimulated cells was carried out with microglia from a single additional case.

#### *4.13. Data Analysis*

Data for relative changes in relation to disease classification were analyzed by one-way analysis of variance (ANOVA) with Newman–Keuls post-hoc test for significance between paired groups. Significant differences were assumed if *p* values of less than 0.05 were obtained. Statistical analyses were carried out using Graphpad Prism Version 7 software (Graphpad Software, La Jolla, CA, USA).

**Author Contributions:** D.G.W. conceptualized this project, planned this study and carried out the immunohistochemistry and biochemical studies with the assistance and input of T.M.T. and L.-F.L. D.G.W. supervised and carried out data analyses and writing of drafts of this manuscript. T.M.T. contributed equally to this project as D.G.W., carried out immunohistochemistry to establish the methodology and initial findings, and carried out all image analyses studies. A.M. carried out confocal immunohistochemistry and microscopy and assisted in image production. I.T. provided funding for studies in Japan and operational support. G.E.S., L.I.S., and T.G.B. selected and provided brain tissues for this study and accompanying clinicopathological data for these cases used from the Brain and Body Donation Program, Banner Sun Health Research Institute. All authors have read and agreed to the published version of the manuscript.

**Funding:** The studies carried out in Japan were supported by grant JP26290022 from Japan Society for Promotion of Science to IT.

**Conflicts of Interest:** The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
