Whole brains perfused with PBS were processed as previously described19 (link). Primary antibodies assessed include: mouse anti-ATXN3 (1H9) (1:1000, MAB5360; Millipore, Billerica, MA), rabbit anti-ASO (1:5000; Ionis Pharmaceuticals, Carlsbad, CA), rabbit anti-NeuN-488 conjugated (1:1000, ABN78A4; Millipore, Billerica, MA), mouse anti-GFAP (1:1000, #3670; Cell Signaling Technology, Danvers, MA), rabbit anti-IBA1 (1:1000, 019-19741, WAKO, Richmond, VA), goat anti-OLIG2 (1:500, sc-19969, Santa Cruz Biotechnology, Dallas, TX), mouse anti-p62 (1:1000, ADI-SPA-902-D; Enzo Life Sciences, New York, NY), and rabbit anti-BECLIN1 (1:1000, ab207612; Abcam, Cambridge, UK). Imaging was performed using an IX71 Olympus inverted microscope (Melville, NY) or Nikon-A1 confocal microscope (Melville, NY) in the basilar pontine nuclei (denoted as pons) and deep cerebellar nuclei (DCN). Nuclear ATXN3 accumulation was quantified as previously described19 (link).
Protocol detail
Immunohistochemical Analysis of Ataxin-3 and Associated Markers
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Antibodies
Beclin1
Brains
Confocal microscope
Deep cerebellar nuclei
Gfap
Goat
Microscope
Mouse
Nuclei
Olig2
Pharmaceuticals
Pons
Rabbit
Corresponding Organization : University of Michigan–Ann Arbor
Other organizations : Ionis Pharmaceuticals (United States)
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Variable analysis
independent variables
- Brain perfusion with PBS as previously described in reference 19
- Nuclear ATXN3 accumulation, as quantified as previously described in reference 19
- Immunostaining for ATXN3, ASO, NeuN, GFAP, IBA1, OLIG2, p62, and BECLIN1
- Whole brains were used
- No positive or negative controls were explicitly mentioned in the provided information.
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