Fusion 2.0 software package

Five randomly selected coronal brain sections, containing both cortex and striatum, from each mouse at 12 months of age were selected. Three mice from each group for BAC-CAG and WT littermates were stained with polyclonal rabbit anti-mouse GFAP primary antibody and goat anti- rabbit secondary antibody, AlexaFluor 488, and counter stained with DAPI. Sections were mounted with anti-fading reagent (ProLong Gold Antifade Mountant, Invitrogen). Images of dorsal striatum and deeper cortical layers with corpus callosum of each brain section were acquired at 40x magnificent using a Dragonfly High-Speed Confocal Microscope 200 and the Fusion 2.0 software package (Andor, Oxford Instrument). Laser and detector settings were maintained constant for the acquisition of each immunostaining. The intensity of GFAP staining of each image was estimated using the Analyze Particles plugin of ImageJ software (NIH). Values from each section were averaged to generate a mean GFAP intensity for each animal.

Immunostaining was carried out using published methods (Gu et al., 2009 (link)). S830 sheep anti-HTT antibody (from Dr. G. Bates; 1:1,000 dilution) antibody was used for mHTT aggregate detection; GFAP antibody (Sigma, 1:20,000 dilution) was used for detection of reactive astrocytes; NeuN antibody (Millipore, 1:1,000) was used to identify neurons; ubiquitin antibody (Dako, 1:1,000 dilution) was used for detection of ubiquitin; ACTN2 antibody (Millipore, 1:500) was used to quantify alpha-actinin 2 expression in striatum. For double immunofluorescent staining (e.g. S830/NeuN) we added the S830 antibody and incubated at 4°C for one day, then added NeuN or other antibodies and incubated at 4°C overnight. Images were acquired using a Dragonfly High-Speed Confocal Microscope 200 and the Fusion 2.0 software package (Andor, Oxford Instrument). Laser and detector settings were maintained at constant for the acquisition of each immunostaining. For all analyses, at least three images were taken per brain region and slide using 360 oil objective lens, at 2,048 × 2,048 pixel resolution, with z-step size of 1 μm at 10 μm thickness.