Immunofluorescence staining experiments and image acquisition and analyses were performed essentially as described (Pak et al., 2015; Patzke et al., 2016). Briefly, cultured neurons were fixed in 4% paraformaldehyde, 4% sucrose in PBS, permeabilized with 0.2% Triton X-100 in PBS, and blocked with 5% goat serum in PBS. Cells were incubated with primary antibodies diluted in blocking buffer overnight at 4°C, washed 3 times, a nd incubated with secondary antibodies in blocking buffer for 1 h at room temperature. Samples were then mounted on glass slides for confocal imaging. The primary antibodies were listed in Key Resources Table. The Alexa-488, Alexa-546-, and Alexa-633-conjugated secondary antibodies were used (Invitrogen). For analysis of neuronal cell body size and total neurite length (Fig. 1B–C), neurons were sparsely labeled with GFP by liposome transfection (Lipofectamin 2000, Life Technologies) with a FUGW construct (Addgene plasmid # 14883) at D9 and fixed at D10 to obtain fluorescent images of individual neurons. To compare the expression pattern of DLK and MKK7 between neurons and glia, co-cultures of human neurons and mouse glia were fixed and immunostained at D12 (Fig. S2B).
