Fig. 3

Imaging of CDK5 substrate phosphorylaiton in primary cells. Rat primary cortical neurons were cultured for 6 days in vitro. a Cells were incubated with 10 μM purvalanol A or vehicle for 3 h prior to fixation, permeabolisation and staining with the indicated antibodies. Phosphospecific antibodies were detected by Cy-3 bound 2ry antibodies (red) and nuclei were counter-stained with the DNA-binding dye DAPI (blue). Scale bar = 60 μm. b The primary neuronal cultures were incubated with either 10 μM purvalanol A, roscovitine or vehicle for 3 h prior to fixation in formaldehyde and embedding in paraffin. Sections were taken from each paraffin block and incubated with the phospho-antibodies as indicated, then biotinylated secondary antibody followed by streptavidin complexed with biotinylated peroxidases which were visualized using DAB staining. Cell nuclei were counterstained with hematoxylin and mounted in DPX. Scale bar = 100 μm. Images are representative of sets from at least three different neuron preparations