Nbp1 05198

Immunostaining of 3D neurospheroids was performed as previously described ^{30 (link)}. Briefly, spheroids were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) overnight at 4°C, washed several times with PBS, and permeabilized in 0.2% Triton-X in PBS for 30 minutes at room temperature. All wash steps and solution changes were accomplished via a two-thirds solution exchange protocol (i.e., aspirate to 25μL/well, add 50μL/well fresh solution). Blocking solution (2% BSA + 0.2% Triton-X in PBS) was then added and spheroids were incubated at 37°C for >1 hour under gentle agitation. Primary antibodies (MAP2, Synaptic Systems 188 004, 1:1000; GFAP, Novus Biologicals, NBP1-05198, 1:1000) were added in blocking solution and incubated overnight at 37°C with gentle agitation followed by 6 washes using PBS + 0.2% Triton-X. Spheroids were blocked again for >1 hour and incubated with secondary antibodies (Invitrogen, A-21450 and A-11039) diluted 1:500 in blocking solution with DAPI (Invitrogen, 1:5000) overnight at 37°C with gentle agitation, protected from light. Spheroids were then washed 6 times using PBS, transferred to flat bottom plates and either stored at 4°C or imaged using an Operetta CLS spinning disk confocal microscope (Perkin Elmer) at 20x magnification (Fig. S1).