Microsuite

Stained slides were evaluated by a trainee (N.H.I.) and a pathologist (A.Y.) on an Olympus BX40 Clinical Microscope and imaging software MicroSuite™ system (Olympus America Inc.). Both assessors were blinded to the treatment groups to minimize operator biasness. A minimum of ten fields were examined and photographed for each tissue section using Olympus DP72 Microscope Digital Camera (Olympus America Inc.). The localisation of the four proteins (Aqp 1, Aqp 5, CFTR and Muc 1) was based on a combinative semi-quantitative H-scoring method (52 (link)). The percentage area of positively labelled cells (value A) and intensity of IHC reaction (value B) were quantified by the operators, and the product of the two values will give a final score (AxB). In the event of discrepancy, slides were re-evaluated to achieve consensus. The scoring for value A is based on percentage area of positively labelled cells (0 = 0%; 1 = below 30%; 2 = 30-60%; 4 = more than 60%). The scoring for value B is based on intensity of IHC reaction (0 = no reaction; 1 = weak reaction; 2 = mild reaction; 3 = strong reaction).

After 3 weeks of treatment with DOCA-salt, the mice were euthanized and the livers were excised, fixed in 4% paraformaldehyde, and embedded in paraffin. The paraffin embedded tissues were sectioned at 4 μm and stained with hematoxylin and eosin (HE) by standard methods. Hepatic steatosis was blindly assessed on 4 random fragments from different areas of each liver and was staged on a scale of 0 to 4, according to the percentage of hepatocytes containing cytoplasmic vacuoles as follows: 0 (<5%), 1 (5–20%), 2 (20%–30%), 3 (30–60%), and 4 (≥60%). Frozen samples were also stained with Oil-Red-O at the optimal cutting temperature. Slides were analyzed by microscopy using Image J and Microsuite (Olympus Soft Imaging Solutions GmbH, Munster, Germany).

Sections of formalin-fixed livers were stained with hematoxylin-eosin and Sirius Red. Frozen samples were also stained with Oil-Red-O that was made at the optimal cutting temperature. Immunohistochemistry staining for cleaved PARP (Abcam, cat. # ab32064), alpha fetoprotein (AFP) (Abcam cat. # ab46799) or sonic hedgehog (shh) (Santa Cruz cat. # sc-9024) was performed in formalin-fixed, paraffin-embedded liver sections according to the manufacturer’s specifications. Slides were analyzed using microscopy using ImageJ and Microsuite (Olympus Soft Imaging Solutions GmbH, Munster, Germany). This histopathology was independently analyzed by a veterinary pathologist (JKP) in a blinded manner (see “Author contributions”).

The livers were fixed in 10% buffered formalin, processed, and embedded in paraffin for hematoxylin-eosin (H&E) staining. Frozen samples prepared at the optimal cutting temperature were also stained with Oil Red O. The microscope slides were analyzed using ImageJ and Microsuite (Olympus Soft Imaging Solutions GmbH, Munster, Germany).
Histological scoring of hepatic steatosis, lobular inflammation, ballooning, and fibrosis was performed by pathologists who were blinded to the group assignments.