Axioplan imaging microscope

Free-floating immunostaining was done following well-established protocols
[10 (link), 11 (link), 34 (link), 35 ]. Sections were incubated in 0.3% hydrogen peroxide in PBS for 20 min, washed in PBS-T (0.01 M phosphate-buffered saline, 0.2% Triton X-100) and blocked 1 h with 10% normal goat serum (NGS) in PBS-T. To examine the evolution of the AD-like amyloid pathology we incubated sections with the monoclonal antibody McSA1
[36 (link)] (MediMabs, Montreal, Canada) at 1:4000 in PBS-T with 5% NGS overnight at 4°C. The following day, the sections were washed in PBS-T and incubated with a goat anti-mouse secondary antibody (MP Biochemicals, Canada) 1:100 in PBS with 5% NGS for 1 h. The sections were washed in PBS and incubated for 1 h with a mouse anti-peroxidase monoclonal antibody
[37 (link)] (1:30) pre-incubated with horseradish peroxidase (5 μg/ml) in PBS (MAP kit, Medimabs, Canada). Stainings were developed with 0.06% 3,3′-diaminobenzidine (Sigma-Aldrich, USA) and 0.01% hydrogen peroxide (Sigma-Aldrich, USA) in PBS and then mounted on subbed slides. Sections were dehydrated in increasing ethanol concentrations (70-100%) and xylene, prior to coverslipping with Entellan (EM Science, USA). Images were acquired on an Axioplan Imaging microscope equipped with an AxioCam HRc digital camera (Carl Zeiss, Toronto, Canada); using the Axiovision 4.8 Software.

Formalin-fixed, paraffin-embedded liver and adipose tissue sections were stained using Hematoxylin and Eosin (H&E). A semi-quantitative scoring system was used to assess hepatocyte steatosis by investigators blinded to the study (0, 1-5% of total area; 1, 5 – 33% of total area; 2, 33 – 66% of total area; 3, >66% of total area [40 (link)]. Adipocyte diameter and surface area were calculated using Adiposoft- ImageJ software.
Oil red O staining of liver sections was performed as described earlier [41 ]. Briefly, cryosections (6-8 microns in thickness) were air-dried and fixed in buffered formalin for 10 min. Sections were washed with water, rinsed in 60% isopropanol and stained with 0.3% Oil Red O solution for 15 min. After rinsing with 60% isopropanol, sections were stained with haematoxylin for 2 min, rinsed in water and mounted using an aqueous mounting media. Photographs were taken using Zeiss Axioplan imaging microscope at 20X magnification.

Immunohistochemistry staining was performed on deparaffinized tongue tissue sections by The Ohio State University Comparative Pathology and Mouse Phenotyping Shared Resource, using Ki-67 antibody (1:100 primary antibody) or antibody against cleaved caspase 3 (1:180 primary antibody). Slides were counterstained with hematoxylin. For Ki-67 staining, cell proliferation was determined by amount of Ki-67 positive nuclei relative to total amount of nucleated cells in six random fields of tongue epithelium using a Zeiss Axioplan imaging microscope at 200× magnification and AxioVision Imaging software (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA).