Imaging and Quantitative Image Analysis

Mosaic images of the immunofluorescently labeled sections were recorded on a Zeiss automatic microscope AxioScan Z1 with high aperture lenses, equipped with a Zeiss Axiocam 506 mono and a Hitachi 3CCD HV-F202SCL camera and Zeiss ZEN 2.3 software. Quantitative image analysis was performed with Image Pro 10 (Media Cybernetics). The cerebral cortex, hippocampus, caudate putamen, and substantia nigra were identified and their size determined by drawing the region of interest (ROI) on the images. Background correction was used if necessary, and the immunofluorescence signal was then macro-based and thus rater-independently quantified by adequate thresholding and morphological filtering (size and shape) to determine the percentage of ROI area covered by immunoreactive (IR) objects, so that the resulting values are normalized for ROI size. After defining the parameters for detection of the targeted objects, image analysis was performed automatically using the same parameters on all images.

The numerical density (n/mm²) of nuclear and cytosolic mHTT inclusions was evaluated in the cortex, hippocampus and caudate putamen. Nuclear mHTT density was evaluated by counting mHTT inclusions overlapping with nuclear DAPI staining whereas cytosolic mHTT density was evaluated by counting mHTT inclusions within NeuN signal that did not overlap with DAPI staining. Data were then normalized by setting the group means of ntg animals to zero.