Fluoview 1000 ix2 inverted confocal microscope

50μm sagittal sections were cut using a Leica VT 1000S vibratome. Epitope retrieval was performed in 10 mM Sodium Citrate 0.05% Tween-20 at 95°C for 15 minutes, and then permeabilized in 1% Triton X-100 in PBS 15 minutes before blocking in 5% normal goat serum. Sections were incubated for 48 hours with primary antibodies 1:500 anti-mouse ubiquitin (ThermoFisher), 1:500 anti-rabbit ATXN1 (12NQ),^{42 (link)}and 1:500 anti-guinea pig CALB1 (Synaptic Systems)) at 4°C. Following incubation, sections were washed four times in PBS and exposed to secondary antibodies (Jackson Immunoresearch Labs Alexa Fluor 488 anti-mouse, Cy3 anti-rabbit, and Alexa Fluor 647 anti-guinea pig) for 24 hours at 4°C. Sections were washed four times in PBS and mounted using ProLong Gold antifade reagent (Thermo Fisher Scientific). Confocal 1.0 micron z-stacks were collected on the Olympus Fluoview 1000 IX2 inverted confocal microscope with a 60x oil immersion objective. Images were merged using Fiji (v2.0.0).