Immunofluorescent staining of undifferentiated (left) and differentiated (right) SH-SY5Y cells. Cells were seeded in a collagen coated 384 well imaging plate at ~ 8,000 cells per well. After overnight incubation, cells were stained using the methanol fix/perm protocol and the anti-MAP2B antibody. Differentiated cells were seeded in a 96 well, collagen coated imaging plate at ~ 5,000 cells per well. Cells were incubated with 50 mM ATRA (Sigma) for 5 days, followed by 50 ng/ml BDNF (Sigma) for 5 days. Differentiated cells were fixed and stained using the methanol fix/perm protocol, and the anti-MAP2B antibody. The second step reagent in both cases was Alexa Fluor® 488 goat anti mouse Ig (Invitrogen). The images were taken on a Pathway 855 or 435 imager using a 20x objective. This antibody also stained undifferentiated SK-N-SH cells using both the Triton X100 and methanol fix/perm protocols.