Scn400f scanner

Whole-slide digital images were provided by the UCL IQpath slide scanning service, using a Leica SCN400F scanner. Representative images of the hippocampus and the PFC were captured with Aperio Imagescope software v12.2.2 (Leica Biosystems, UK) at ×2 magnification.

Treatment- and test-naive C57BL/6J males and females were used. Mice received a single dose of Raphin1 or cycloheximide (CHX, Sigma-Aldrich) at 40 mg/kg at 3 months of age or were treated chronically with Raphin1 at 2 mg/kg from 4 weeks of age for 10 weeks before the analysis. Mice were culled by cervical dislocation, liver was extracted, fresh-frozen in the O.C.T. compound (VWR International) and kept at −80°C until use. Oil Red O staining was performed on 10 μm thick frozen cryosections (Leica CM1850). Sections were washed in 60% propanol and stained for 20 min at room temperature with filtered 0.33% Oil Red O solution (VWR International). After rinsing with distilled water sections were counterstained with hematoxylin (VWR, diluted 1:15) for 1 min. Images were acquired with a 40X objective using SCN400F scanner (Leica).

Half brains were immersion fixed in 10% buffered formal saline (Pioneer Research Chemicals) for a minimum of 48 h prior to being processed to wax (Leica ASP300S tissue processor). The blocks were trimmed laterally from the midline by ~ 0.9–1.4 mm to give a sagittal section of the hippocampal formation. Two 4 μm sections 40 μm apart were analysed. The sections were pretreated with 98% formic acid for 8 min, followed by washing. The slides were wet loaded onto a Ventana XT for staining (Ventana Medical Systems, Tuscon, AZ, USA). The protocol included the following steps: heat induced epitope retrieval (mCC1) for 30 min in Tris Boric acid EDTA buffer (pH 9.0), superblock (8mins) and manual application of 100 μl of directly biotinylated mouse monoclonal IgG1 antibodies against Aβ (82E1, IBL, 0.2 μg/ml or 4G8, Millipore, 2 μg/ml) for 8 h. The staining was visualised using the Ventana DabMap kit (iView DAB, Ventana Medical Systems), followed by 4mins of haematoxylin and blueing. Alternatively, for staining of Beta-amyloid, slides were incubated with mouse monoclonal 6F/3D (Dako 1:50) followed by Iview Ig secondary antibody (Ventana Medical Systems). The sections were dehydrated, cleared and mounted in DPX prior to scanning (Leica SCN400F scanner). All images were analysed using Definiens Tissue Studio software (Definiens Inc).

The right and left patellar tendons were harvested from two of the intact Macropus rufogriseus (specimens 1 & 2). The tendons dissected from the thawed cadavers were fixed in 10% neutral buffered formalin. Samples containing bone (diagnosed via imaging, above) were decalcified in 10% formic acid solution. All specimens were sectioned in the sagittal plane along the midline and directly lateral to the midline. The tendon sections were dehydrated and embedded in paraffin wax blocks. Microtome sections were cut between 4 and 6 µm. Sections were stained with Haematoxylin and Eosin, and Masson’s trichrome. In the case of tendons where ossified patellar tissue was found to be present, sections were also stained with Safranin O/Fast Green for the identification of cartilage, and Von Kossa to highlight the presence of calcium salts. The histological sections were examined by light microscopy and images obtained via scanning at high resolution using a Leica SCN400F scanner.

Nerve biopsies were processed according to local standard operating procedures. In brief, fresh biopsies, immediately after removal, were divided in half, with one portion fixed in 10% buffered formalin, followed by tissue processing and embedding in paraffin and the other portion fixed in 3% glutaraldehyde, followed by processing into epoxy resin. The paraffin embedded tissues were cut at 5-µm thin sections and stained with a battery of histochemical and immunohistochemical stains (including immunostaining for neurofilaments with SMI31 antibody (1:5000; Sternberger, shown in Fig. 3) and the resin-embedded tissues were cut into 1-µm semi-thin sections and stained with methylene blue azure-basic fuchsin. All slides were viewed under light microscope. Histology images shown in the Fig. 3 were digitized on a LEICA SCN400F scanner at ×40 magnification and 65% image compression setting during export. For the preparation of light microscopy images, image captures were taken in LEICA SlidePath (LEICA Milton Keynes, UK). Publication figures were assembled in Adobe® Photoshop.
For the analysis of myelinated fibre density in sural nerve biopsies, four randomly selected fascicles from each case were assessed. Both large and small myelinated fibres were counted.

IHC was performed on formalin-fixed paraffin-embedded sections using standard protocols. For collagen staining, sections were rehydrated and then immersed in Picro Sirius red solution (0.1% Direct red 80, Sigma 41496LH and 0.1% Fast green FCF, Raymond Lamb S142-2) for 2 h.
Slides were imaged using the Leica SCN 400f scanner and analyzed using Leica Slidepath Digital Hub software.