Immunofluorescence staining (IF)—human tissue
For all qualitative IF analysis of human brain tissue samples, freshly cut sections were prepared from late stage (Braak V–VI) AD and control patient tissues archived at the UNC Brain Bank. Paraffin embedded tissue blocks were sectioned at 7 µM thickness, adhered to slides, and deparaffinized in xylene. Slides were rinsed in ethanol and rehydrated (pure, 95%, 70%) in 1 X PBS. Sections were boiled for 10 min and incubated in hot Vectastain H-3300 citrate-based buffer for 30 min for antigen retrieval. Sections were then rinsed in 1X TBS, and permeabilized for 50 min in 2% TBS-Triton X-100 at RT. Blocking, primary, and secondary solutions matched the donor species of secondary antibodies used; sections were blocked in 1X TBS with 2% goat serum for 50 min. Sections were then incubated with primary antibody in 0.1% sodium azide solution for 48 h at RT, thoroughly rinsed in 1X TBS three times, and incubated in secondary antibody solution for 24 h at RT. DAPI staining was used to visualize nuclei. Primary antibodies and dilutions: Tau-1 (Millipore, MAB3420, 1:1000), GFAP 1:1000, (Dako, GA524). Secondary antibody dilutions: Alexa Fluor goat anti-mouse IgM μ-chain specific 488, 1:200 (Thermofisher, A-21042), Alexa Fluor goat anti-rabbit 568, 1:200 (Thermofisher, A-11011), Alexa Fluor goat anti-mouse IgG1 specific 647, 1:200 (Thermofisher, A-1240).
Immunofluorescence Staining (IF)—mouse tissue
Mice were deeply anesthetized and transcardially perfused with 1X PBS and 15 mL 4% Paraformaldehyde. Brains were then post-fixed for 24–48 h in 4% PFA at 4 °C and cryoprotected in 30% sucrose solution before sectioning. Tissues were sectioned at 30–40 μM thickness and stored at −20 °C. Immunofluorescence staining was conducted using a free-floating technique, rinsing in 1X TBS between all major steps, gently agitated on a plate shaker. Sections were rinsed and permeabilized for 50 min in 2% TBS-Triton X-100 at RT. Blocking, primary, and secondary solutions matched the donor species of secondary antibodies used; sections were blocked in 1X TBS with 2% goat serum for 50 min each. Sections were then incubated with primary antibody in 0.1% sodium azide solution for 48 h at room temperature, thoroughly rinsed, and incubated in secondary antibody solution for 24 h at room temperature. DAPI staining was used to visualize nuclei. Primary antibodies and dilutions: Mouse IgM, kappa monoclonal MM-30, 1:1000 (Abcam, ab18401), AT8 1:500 (Thermofisher, MN1020), GFAP 1:1000, (Dako, GA524). Secondary antibodies and dilutions: Alexa Fluor goat anti-mouse IgM μ-chain specific 488, 1:200 (Thermofisher, A-21042), Alexa Fluor goat anti-rabbit 568 1:200 (Thermofisher, A-11011), Alexa Fluor goat anti-mouse IgG1 specific 647, 1:200 (Thermofisher, A-21240).
Immunohistochemical Staining (IHC)—Human tissue
For all IHC analysis of formalin fixed, paraffin embedded tissues (n = 124 total cases), brain samples were acquired from the Duke Bryan Brain Bank (Table 1), and samples were sectioned at 8 µm thickness and adhered to slides. Tissue sections were deparaffinized with xylene and rinsed with ethanol (pure, 95%) prior to staining. Endogenous peroxidase activity was blocked via incubation in 1.875% H2O2 in methanol for 8 min. Sections were rinsed with 1X PBS, and blocked with 5% non-fat dry milk in 0.05 M Tris buffer, Ph7.6 for 20 min at room temperature. Sections were rinsed in deionized water three times for two minutes each. Sections were incubated for 45 min at 37 °C with AT8 (Thermofisher, MN1020, 1:500) primary antibody, rinsed three times in deionized water, and incubated for 30 min with Dako EnVision Dual Link System-HRP at 37 °C. Sections were again rinsed in deionized water three times before development with Dako DAB Solution (Agilent, K346811-2) for 5 min before rinsing in tap water for 5 min. Counterstain was applied with Fisherfinest Hematoxylin + (220-100) for 25 s before an additional tap water rinse for 5 min. Blueing of sections was achieved with ammoniacal water for 10 s before a final 3-min rinse in tap water. Sections were dehydrated in serial graded ethanol rinses, cleared in xylene and coverslipped with permount before scanning and analysis.
Analysis of IHC sections
Neuropathological evaluations were performed by the Duke Bryan Brain Bank, including Braak stage, AD composite neuropathological score, age, postmortem interval, cognitive status, sex, ethnicity, and APOE allele status (Table 1). Composite neuropathological scoring was assessed according to guidelines in Montine et al. . Tissue sections were immunostained with AT8 for tau pathological staging as previously described  and counterstained with hematoxylin to identify CA structures. All slides were scanned at the UNC Translational Pathology Laboratories on a brightfield Scanscope AT2 at 40× magnification. Images were inspected for quality and cropped to the hippocampal region in Aperio Imagescope; within this image, the dentate gyrus was traced as the analysis region of interest, and area measured for CA density calculation. CA were annotated and counted by three independent investigators blinded to patient status. To distinguish CA from smaller nuclei within regions of interest, criterion for CA detection included low to moderate hematoxylin staining intensity with a uniform or concentric ring pattern and circularity.
Statistical analysis—PAS granules in tauopathy and APOE-KI mice
The WT and PS19 animals were compared in terms of PAS granule density, GFAP Intensity, and AT8 intensity using an unpaired t-test, with a 5% significance level. The overall difference in PAS granule density between APOE-KI mice (E2, E3, E4) was assessed using an unpaired t-test, with 5% significance level. Stratified analysis by sex was also performed.
Statistical analysis—CA quantification
Summary statistics including mean, standard deviation, median, interquartile range, and range were computed for the continuous variables. Absolute and relative frequencies were calculated for the categorical variables. Box plots and scatter plots were constructed to assess the association between CA and Braak stage. Plots using the log scale for the CA counts are also provided. The association between CA and Braak stage was then evaluated using a generalized linear model with gamma distribution and log link function, controlling for age (years), PMI (minutes), cognitive status, sex, and APOE allele. Results are presented as mean ratios (MR) with confidence intervals (CI). Multiple comparisons among Braak stages are performed using the Bonferroni method. All statistical analyses were performed in R version 4.0.2 (R Core Team, 2020). Complete case analysis was considered, with P < 0.05 determining statistical significance.
Immunofluorescence staining (IF)—human CSF
For qualitative IF analysis of CA present in CSF, human control and AD patient CSF samples (n = 2 patient samples for each) were obtained from the University of Miami Brain Bank, aliquoted, and stored at -80 °C. Aliquots (200 μl each) were centrifuged three times for 10 min at 700 g in 4 °C before fractionation for amyloglucosidase digestion and resuspended with 1X PBS each time. Digested fractions were incubated with 10U amyloglucosidase (Sigma, 9032-08-0) in phthalate buffer (pH 5) for 24 h at 45 °C. Undigested fractions were incubated at 4 °C for 24 h in phthalate buffer without amyloglucosidase. After digestion/incubation, fractions were centrifuged three times for 10 min at 700 g in 4 °C and resuspended with 1X PBS each time. Following the final centrifugation, samples were extended on charged Superfrost slides, air dried overnight, and fixed with acetone for 10 min at 4 °C. Slides were then rinsed with 1X TBS three times for 5 min, before being permeabilized with 1X TBS + 2% Triton X-100 for 30 min at RT. Slides were incubated with blocking buffer (1X TBS + 2% Goat Serum) for 30 min at RT. Digested and undigested fractions were incubated overnight at RT in blocking buffer containing Tau5 (Ms IgG1, MAB361, Millipore) at 1:200. Secondary only controls were instead incubated in empty blocking buffer. All samples were then incubated overnight at RT in secondary antibody solution (blocking buffer) with Alexa-Fluor anti-mouse IgM 488 (Thermo, A-21042) at 1:200 and Alexa-Fluor anti-mouse IgG1 (Thermo, Cat A-21240) at 1:200. Slides were coverslipped in Fluoromount-G and dried overnight before imaging at 60× magnification on an Olympus Fluoview FV3000RS Confocal laser scanning microscope. Since this was a qualitative analysis performed by immunofluorescence microscopy (representative images are depicted in Fig. 4C), additional sample numbers will be required for future statistical comparisons between sample groups (e.g., control vs. AD).
Confocal images were captured on an Olympus FV3000RS microscope using resonant, one-way scanning. The following fluorophores were used: Alexa Fluor 405 (DAPI), Alexa Fluor 488, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 647. Laser intensities, gains, and offsets were set at thresholds that reflect minimal fluorescence in respective control stains of sections incubated with only blocking buffer and secondary antibodies for each experiment. Imaging parameters were tailored for each experimental application, but were kept consistent between genotypes, adsorption pairs, and all other respective experimental and control stains; sections used for quantification of PAS granules were imaged at 20× magnification with 2 × zoom. For co-localization analysis, human tissue sections were imaged at 20× magnification with 3 × zoom. Phase separation ensured that fluorescence spectra did not significantly overlap (pairing: 405 + 568/594, 488 + 647, when applicable). High-resolution images for analysis of CA in CSF were captured using 60× magnification and 2 × zoom.