Immunocytochemical/Immunofluorescence analysis of mouse primary neurons stimulated with 5 μM of MPP+ for 0, 6, 12, and 24 hours labelling BPOZ (green), TH (red) and Nuclei (DAPI - blue). Coverslips containing neurons cultured to 70–80% confluence were fixed with chilled methanol overnight, followed by two rinses with PBS. Samples were blocked with 2% BSA in PBS containing Tween 20 and Triton X-100 for 30 minutes and incubated at room temperature under shaking conditions for 6 hours in PBS containing the ab1077 (1:200), TH (1:1000). After four 15 minute washes in PBS, slides were incubated with Cy2- or Cy5-labelled secondary antibodies (1:200) for 1 hour under shaking conditions. Following four 15 minute washes with PBS, cells were incubated for 4–5 minutes with DAPI (1:10000).
