Pannoramic 250 digital slide scanner

We used EM48 antibody to detect mHtt aggregates, which targets the N-terminal region of human Htt (amino acids 1-212). The analysis was performed on the paraffinized brain Sections (10 µm) with the ABC method. After deparaffinisation, antigen retrieval and endogenous peroxidase inactivation, sections were incubated in TBS-T (1×TBS, 0.1% Tween20). Nonspecific binding was blocked by BSA (4% BSA, 0.3% triton-×-100, PBS). The sections were incubated with mouse EM48 (1:100, Millipore #MAB5374) for 48 h at 4 °C. After several washes in TBS-T (1×TBS, 0.1% Tween-20), the sections were incubated with goat anti-mouse secondary antibodies (1:200, Vector Laboratories, Burlingame, CA), followed by incubation for 30 min with ABC complex. Staining was developed with the DAB method with DAB substrate kit (SK-4100, Vector), then sections were dehydrated and mounted using cover slips. All sections were scanned with a Pannoramic 250 digital slide scanner (3DHISTECH, Hungary). Three to six striatal brain sections were used to measure immunoreactivity, which was quantitated relative to the control group (N171-82Q-PBS). Healthy animals (WT) did not have mHtt aggregates and were not used in the analysis. Aiforia Create version 5.0 (Aiforia Technologies Oy) was used to analyse EM48-immunoreactivity and inclusions.

Femurs resected from control mice and LLC-bearing mice on day 25 postinoculation of LLC cells and sham operation, respectively, were fixed with 4% PFA (3M Science) for 7 days. Then, the femurs were decalcified in 14% EDTA (pH 7.0) and embedded in paraffin. Then, 4.0-micrometer-thick sections were prepared using Leica RM2255 microtome (Leica Biosystems, Wetzlar, Germany) and used for TRAP staining (GENOSS, Suwon, Korea) and hematoxylin counterstaining (GENOSS, Suwon, Korea). The stained sections were observed and images were captured using Pannoramic 250 digital slide scanner (3D Histech, Hungary). TRAP-positive multinucleated osteoclasts in the metaphyseal area, located 0.5 mm proximal to the growth plate and 0.5 mm away from the intracortical surface, were counted and the number of TRAP-positive multinucleated osteoclasts normalized to the bone perimeter were analyzed for each sample. The bone perimeter was measured using ImageJ software v 1.53e (NIH, Bethesda, MD, USA).