For IHC, thin-layer 3D cultures were permeabilized and blocked by incubating with the blocking solution at 4°C for 12 hours. To block endogenous peroxidase activities, the cultures were incubated with 0.05% (v/v) H2O2 solution in TBS for 5 min at room temperature, washed with TBST 3 times and incubated with the blocking solution for 2 hours at room temperature. After incubating with the primary antibody solutions for 24 hours at 4°C, the cultures were washed 5 times with TBST and then incubated with ImmPRESS anti-mouse or -rabbit Ig (ImmPRESS Peroxidase Polymer Detection Kit, Vector Laboratories, Burlingame, CA, USA) for 30–60 minutes. The cultures were washed 5 times for 10 min each with TBST and developed by using ImmPACT DAB Peroxidase Substrate kit (Vector Laboratories). The following antibodies and dilution rates have been used in this study: BA27 anti-Aβ40 antibody HRP-conjugate (1:2, Wako Chemicals USA, Richmond, VA, USA), BC05 anti-Aβ42 antibody HRP-conjugate (1:2, Wako Chemicals USA), AT8 anti-p-tau antibody (1:40, Thermo Scientific), PHF1 anti-p-tau antibody (1:1,000), anti-MAP2 antibody (1:200, Cell Signaling Technology), ImmPRESS anti-mouse and -rabbit Ig HRP polymer conjugates (1:2, Vector Laboratories).
