Triton x 100

Rat primary cortical cultures were established in black-walled, glass bottom 96-well plates (μClear, Greiner Bio-One) with the same cell density and culture conditions as described for MEA experiments. Following a 4-day treatment with extract from AD patient, ALB01, or vehicle (media alone), the cultures were washed with ice cold PBS and fixed with 4% paraformaldehyde in PBS. Following fixation, the cells were permeabilized with 1% Triton X-100 in donkey serum (Jackson ImmunoResearch). The cells were probed with primary antibodies for tau (1:200, A0024, Dako), followed by anti-rabbit Cy3 secondary antibodies (Jackson ImmunoResearch) and 1 μg/mL DAPI (Thermo Fisher). Mouse anti-Vglut2 (Abcam, 1:1000) and rabbit anti-HOMER1 (Synaptic Systems, 1:1000) were used to quantify VGLUT2 and HOMER1 in aged (Day 21) iNs transduced with the lentivirus (Lenti-eGFP or Lenti-GM2A) at MOI = 5 on Day 5 or treated with TBS or AD brain extract (ALB01) from Day 17. Analysis was performed using Fiji (National Institutes of Health) by counting the number of puncta along each neuritic segment (~ 10 um), and 15 images were counted in each condition. Co-localization of puncta was defined by overlap within a circular area covering 0.52 um². Images were acquired at 40X using an LSM710 confocal microscope (Zeiss).

The assay was performed as previously described (Houlihan et al., (link)). Briefly, paraffin‐embedded kidney slides were deparaffinized and rehydrated with xylene, ethanol and water sequentially. Antigens were unmasked by incubating slides in an Antigen unmasking solution (H3300, Vector lab) and boiling for 20 min. Non‐specific binding was blocked by incubation with 0.05% Triton‐X 100 and 5% normal goat serum (005‐000‐121, Jackson ImmunoResearch) in PBS at room temperature for 1 h. Slides were then incubated with a rabbit antibody against cleaved caspase‐3 (9661, Cell Signaling Technology) and mouse antibody against β‐catenin (610153, BD Transduction Laboratories) and Hoechst 33342 for staining nuclei (Molecular Probes) at 4°C overnight. An Alexa Fluor 488 AffiniPure Goat ant‐rabbit IgG (111‐545‐144, Jackson ImmunoResearch) and a Cy3 AffiniPure Goat anti‐mouse IgG (115‐165‐146, Jackson ImmunoResearch) were used to recognize the respective anti‐cleaved caspase‐3 and β‐catenin antibodies.

Immunohistochemistry was performed on 20‐μm thick cryosections (for ages up to P3) or 50‐μm thick free‐floating sections. Sections were washed with 0.05 M PBS and incubated with a blocking solution containing 5% normal donkey serum (NDS, Jackson ImmunoResearch), 2% bovine serum albumin (BSA, Sigma), 0.2% Triton X‐100 (Sigma) in PBS (22–24 °C, 1 h). Next, tissues were exposed to a solution containing 2% NDS, 0.1% BSA, 0.2% Triton X‐100 in PBS and a pre‐defined mixtures of primary antibodies (Table 4) at 4°C for 72 h. After extensive rinsing in 0.05 M PBS, secondary antibodies conjugated to cyanine (Cy)2, Cy3.,or Cy5 (1:300, made in donkey; Jackson ImmunoResearch) were applied (22–24°C, 2 h). Hoechst 33342 (1:10000, Sigma, #23491‐52‐3) was routinely used as a nuclear counterstain. After repeated washes in PBS, sections were dipped in distilled water, mounted, air‐dried, and coverslipped with Entellan (in toluene; Merck).