Immunofluorescent staining of differentiated SH-SY5Y cells (right). Cells were seeded in a 96 well, collagen coated imaging plate at ~ 5,000 cells per well. Cells were incubated with 50 M ATRA for 5 days, followed by 50 ng/ml BDNF for 5 days. Differentiated cells were fixed and stained using the Triton X100 fix/perm protocol (see Recommended Assay Procedure, Bioimaging protocol link) and the anti-SMN antibody. The second step reagent was Alexa Fluor® 488 goat anti mouse Ig (Invitrogen)(pseudo colored green). Cell nuclei were counter stained with Hoechst 33342 (pseudo colored blue). The image was taken using a 20x objective and merged using the BD AttoVison ™ software This antibody also stained undifferentiated SH-SY5Y, SK-N-SH, C6, U87 and U373 cells using both the Triton X100 and methanol fix/perm protocols (see Recommended Assay Procedure, Bioimaging protocol link).