Streptavidin biotin blocking kit

Mouse brains were hemi-sectioned and halves placed directly into 10 ml of Formalin-Free Tissue Fixative (Sigma, St. Louis MO) and stored at 4 °C until prepared for sectioning. To assess the extent of brain neuropathology, paraffin-embedded hemibrains were serially sectioned at 10 µm intervals. Twelve matched slices from each brain were taken from equivalent regions (plates 12–16, ref.⁵⁰ ), deparaffinized and processed for amyloid detection. For immunohistochemistry, sections were washed in Tris-buffered saline (TBS), blocked with a streptavidin/biotin blocking kit (ThermoFisher Scientific, Waltham, MA), and then incubated in TBS containing 0.5% Tween and 5% donkey serum for 30 min. Tissues were incubated in 4G8 antibody (1:1000; Biolegend) diluted in blocking solution at 4 °C overnight. After washing, sections were incubated in biotinylated secondary antibody (Jackson Immunoresearch; dilution 1:200), followed by incubation with Dylight 549 Streptavidin. Fibrillar Aβ deposits were visualized using Thioflavin S (Thio-S), following previously described methods^{51 (link)}. Briefly, mouse brain sections were washed with TBS and stained for 10 min with a solution of 0.5% Thio-S in 50% ethanol. Finally, sections were washed in 50% ethanol and TBS, dried, and coverslipped using Vectashield (Vector Laboratories, Burlingame, CA).

miniSOG⁺ HEK293T cells or neurons were fixed and glycine-quenched as described above, then permeabilized in 0.1% Triton X-100 in PBS for 15 min, blocked with a streptavidin/biotin blocking kit according to manufacturer’s instructions (Thermo Fisher Scientific, cat. no. E21390), and then polymerized as described above for DAB in the presence of 1 mM biotin-DAB instead for 10 min at ~155 mW/mm² of a 475-nm light. The same was true for neurons, except that the intensity was reduced to ~3.2 mW/mm². Following polymerization, cells were washed 3× for 5 min with PBS + 0.1% Tween 20 and then stained with a 1:750 dilution of streptavidin-AF647 (Thermo Fisher Scientific, cat. no. S21374) in PBS/Tween 20 for 90 min, followed by 3× 20-min washes of PBS/Tween 20 before confocal imaging.