Ventana ultra system

After formalin fixation, dehydration, and paraffin embedding, 4 µm sections were stained for Hematoxylin Eosin (HE), PicroSirus red (PSR), and immunohistochemistry (IHC). IHC was performed on an automated Ventana Ultra system (Ventana Medical Systems, Roche Group, USA). The rat monoclonal (M3/38) antibody against MAC2 (Galectin-3, CL8942AP, Cedarlaine, 1:5000 dilution) or rabbit polyclonal antibody against COL1A1 (LS-C343921, LSBio, 1:1000 dilution) were used to detect macrophages or collagen deposits in liver sections, respectively. Antibody binding was visualized using DAB, hematoxylin was added as a nuclear counterstain.
Image analysis was performed on digital images of the HE, MAC2, and COL1A1 stained slides, using Visiopharm Integrator System software (v2019.2, Visiopharm). Lipidosis was calculated based on the unstained area (fat) on the HE stained slides and corrected to the total section area. For MAC2 and COL1A1 quantification, DAB positive area was quantified and normalized to the total section area. The areas were detected by threshold and machine learning.
Fibrosis score assessment was performed on PSR stained slides at 200x magnification, according to Kleiner et al.^{56 (link)}. The definitions of fibrosis stages are: 0= no fibrosis, 0.5-1= perisinusoidal or periportal, 2= perisinusoidal and portal/periportal, 3= bridging fibrosis, 4= cirrhosis.

Snap-frozen liver samples were cut (6 µm thick) and stained with ORO according to standard procedures. After formalin fixation, dehydration and paraffin embedding, 4-µm sections were stained with haematoxylin and eosin and picrosirius red according to standard procedures. Consecutive sections were immunohistochemically stained for Mac2 (CL8942AP; Cedarlaine) and Col1a1 (LS-C343921; BioSite) in an automated Ventana Ultra system (Ventana Medical Systems). Image analysis was performed on digital images using Visiopharm Integrator System software (v.2018.09). Lipid-filled vacuoles, liver steatosis, inflammation, the NAS and the fibrosis stage were evaluated in the haematoxylin and eosin- and picrosirius red-stained liver sections according to the methods reported by Kleiner et al^{28 (link)}. All histological assessments were performed blind by a board-certified veterinary pathologist.