Immunocytochemistry and optical microscopy

For immunocytochemical analysis of DCC, cells on coverslips were fixed with 4% paraformaldehyde (Wako, Osaka, Japan) in Krebs’ buffer with 0.4 M sucrose for 20 min at room temperature [15]. Fixed cells were treated with 0.1% Triton X-100 for 5 min, and then incubated with an anti-DCC antibody (1 μg/mL) for 1 h. DyLight 488-conjugated donkey anti-mouse IgG (H + L) (Jackson ImmunoResearch, West Grove, PA) was used at 17.5 μg/mL to visualize total DCC. After mounting the coverslips in a drop of ProLong Gold mounting medium (Thermo Fisher Scientific Inc., Waltham, MA), twelve-bit grayscale images of neurons were collected using an inverted fluorescence microscope (model TE2000-U; Nikon, Tokyo, Japan) equipped with 60×/1.45 NA oil-immersion and 40×/0.75 NA dry objectives, and a cooled charge-coupled device (CCD) camera (model ORCA C4742-95-12NR; Hamamatsu Photonics, Shizuoka, Japan).

For morphometric analysis, cortical cultures were treated with 250 ng/mL netrin-1 (R&D Systems, Minneapolis, MN) for 30 min or 4 h by bath application [11, 15] in the presence or absence of 1 μg/mL anti-DCC function-blocking antibody that was bath-applied 30 min earlier than netrin-1 application [33], then fixed. In the initial several rounds of the experiments, surface L1 was visualized to distinguish the axons from the dendrites, since L1 is known to accumulate at the axonal surface [35]; for this purpose, fixed cells were incubated with an anti-L1 antibody (5 μg/mL) for 16 h without permeabilization, and then incubated with Cy3-conjugated donkey anti-rat IgG (H + L) (15 μg/mL; Jackson ImmunoResearch) for 1 h. After collecting DIC and phase contrast images (as well as epifluorescence images in case of surface L1 immunocytochemistry), we employed DIC images for morphometric analysis.