Slides were prepared as described above for the PD30 animals (25-μm sections). The samples were washed twice for 10 minutes in 1X PBS then permeablized for 20 minutes in 0.1% Triton X-100 in PBS. Following 3 washes in PBS, the sections were incubated with 1% BSA in PBS for 3 hours. The BSA solution was removed and the sections were incubated overnight with primary antibodies. The antibodies and their dilutions were as follows: rabbit anti-β-gal (1:100; MP Biomedicals, Ohio, USA), mouse anti-neuron-specific nuclear marker (NeuN; 1:100; Millipore, Massachusetts, USA) and chicken anti-glial fibrillary acid protein (GFAP; 1:500; Millipore). The dilutions were made with 1% BSA in PBS. After antibody incubation, the slides were washed 5 times for 6 minutes with 1X PBS followed by a 2-hour incubation with a 1:100 dilution of each secondary antibody. The following antibodies were used (all from Invitrogen, California, USA): Alexa 488 (goat anti-rabbit), Alexa 350 (goat anti-mouse IgG1) and Alexa 594 (goat anti-chicken). The sections were washed 5 times for 10 minutes with 1X PBS and mounted with Vectashield (Vector Laboratories, California, USA). Microscopy was done as described above.
