Immunohistochemistry and stereological measurements

Mice were deeply anaesthetized and transcardially perfused with 4% paraformaldehyde in PBS. Brains were processed for paraffin embedment. Brain sections (5 μm, coronal) were used for immunohistochemical localization of DARPP-32 (1:500, Epitomics) using the IHC Select HRP/DAB kit (Millipore). Quantitation of DARPP-32 immunostaining was conducted using NIH image J software. The same image exposure times and threshold settings were used for sections from all treatment groups.

To measure the number of NeuN-positive cells, a series of 25 μm thick coronal sections spaced 200 μm apart spanning the striatum were stained with NeuN antibody (Millipore, 1:500) and visualized by diaminobenzidine. For neuropathological analyses, brain sections were analysed stereologically. Briefly, unbiased stereological counts of NeuN-positive neurons within the striatum were performed using unbiased stereological principles and analysed with StereoInvestigator software (Microbrightfield, Williston, VT). Optical fractionator sampling was carried out on a Leica DM5000B microscope (Leica Microsystems, Bannockburn, IL) equipped with a motorized stage and Lucivid attachment ( × 40 objective). The following parameters were used in the final study: grid size, (X) 500 μm, (Y) 500 μm; Counting frame, (X) 68.2 μm, (Y) 75 μm, depth was 20 μm. Gundersen coefficients of error for m=1 were all less than 0.10. Stereologic estimations were performed with the same parameters in striatum of wt or YAC transgenic mice treated with the control peptide or peptide HV-3 (n=6 mice per group). The total volume of stratial tissue measured in each brain is calculated by StereoInvestigator and the neuronal density is presented as NeuN positive cell number per mm³.

Quantitation was conducted by an experimenter blind to the experimental groups.