Histology

Tibialis anterior (TA) from male mice for the four genotypes (n = 3 per group) were dissected at 12 week of age, weighed and snap frozen and 10 µm sections were cut in a cryostat. TA sections were stained for NADH-tetrazolium reductase and ATPasa.

To detect mutant human huntingtin intranuclear inclusions in the brain, we conducted the same protocol as previously described (29). Mice were transcardially perfused with 4% paraformaldehyde (PFA), brain removed and placed in PFA for another 4–6 h. After 30% sucrose protection, brains were crypreserved at −80° until used. Free-floating cryosections at 30 µm were immunostained against mutant huntingtin using mouse anti- human polyQ (MAB5374 Millipore, 1:500) overnight. The seconday antibody was Alexa 488 (Life Technology) and nuclear DAPI counterstaining (Prolong, Life technology). Confocal images were taking using Zeiss LSM 700 microscope at 60× amplification of the granular layers in the cerebellum. Final images stack projections (typically the sum of 8 images per projection) were used to count the total of inclusions per field. A minimum of three fields per slice, over 3 or 4 slices were used to account for the total number of inclusions per mouse. Volocity software 5.4.1 (Perkin Elmer, USA) was used to analyse the total count and classified by the size of the inclusions per image. The given data is a relative proportion of each size group to the total, falling into four categories, from the smallest aggregates (0–2 µm²) to the biggest inclusions found (>6 µm²).