Immu mount solution

The SH-SY5Y cells were fixed with 4% paraformaldehyde in PBS and blocked with a blocking solution (5% normal goat serum [Invitrogen], 0.1% Triton X-100 [Sigma-Aldrich, St. Louis, MO, USA]) in PBS, pH 7.4) for 1 h at room temperature and incubated with primary antibodies against HA-tagged β23 at 4 °C overnight. Then, the cells were washed with wash buffer (0.1% Triton x-100 in PBS, pH 7.4) and incubated with corresponding secondary antibodies conjugated with fluorescent dyes at room temperature for 1 h. After simple washing with wash buffer, the samples were incubated with DAPI (Sigma-Aldrich, St. Louis, MO, USA) solution. The cells were washed with DAPI solution in PBS and mounted on a microscope slide using Immu-Mount solution (Thermo Fisher Scientific, #9990402). Images were obtained using a fluorescent microscope (Axio Imager M2; Carl Zeiss, Oberkochen, Germany).

For immunostaining, 0-, 3-, 7-, 10- and 14-day GFBL cultures generated on gelatin coated glass coverslips (with cells) and parallel CDMs were washed once with PBS, fixed with 4% formaldehyde at room temperature for 20 min, washed three times with PBS, and stored at 4 °C in PBS until immunostained. For immunostaining, cells were permeabilized with 0.5% Triton X-100 in PBS for 4 min. Samples were then washed three times with PBS followed by blocking with PBS + (containing Ca²⁺ and Mg²⁺) containing 10 mg/mL BSA and 1 mg/mL glycine at room temperature for 30 min. Samples were then incubated with a primary antibody (Supplementary Table S2) diluted in 1 mg/mL BSA in PBS in a humidified chamber at 4 °C overnight. The samples were then washed with PBS containing 1 mg/mL BSA and 0.01% Triton X-100 and then incubated with an appropriate Alexa-conjugated secondary antibody (1:200 dilution; Alexa 488/594; Thermo Fisher) at room temperature for 1 h. Nuclei were then stained with 300 nM DAPI (Thermo Fisher) in PBS for 10 min. Samples were mounted with Immu-Mount™ solution (Thermo Fisher) and examined using a Nikon Eclipse 80i Compound Fluorescent Microscope (Nikon Corporation, Tokyo, Japan) and images were captured using Nikon NIS-Elements Basic Research 4.2 software (Nikon Corporation).