*2.3. Histology and Immunohistochemistry*

Paraffin-embedded liver samples were cut into 3–6 μm sections and routine hematoxylin/eosin and Masson's trichrome staining were performed. For Sirius Red/Fast Green staining, sections were deparaffinized, hydrated, incubated in a staining solution consisting of 0.1% Sirius Red (Polysciences, Inc., Warrington, PA, USA) and 0.1% Fast Green (Roth, Karlsruhe, Germany) in saturated picric acid (Chroma, Münster, Germany) for one hour and differentiated in 1% acetic acid for 45 seconds.

To perform immunohistochemical stainings, peroxidase activity was blocked with 3% hydrogen peroxide. Afterwards, sections were boiled for 10 minutes either in citrate buffer (pH 6.0, Collagen I), Tris-EDTA buffer (pH 9.0, CD45) or no target retrieval was performed (α-SMA). Sections were then blocked with 10% BSA (PAA, Pasching, Austria) and 2.5% normal horse serum (Vector Laboratories, Inc., Burlingame, CA, USA) and incubated with specific antibodies (Rabbit anti Collagen I polyclonal antibody, ab34710, Abcam, Cambridge, UK; Rabbit anti CD45 polyclonal antibody, 20103-1-AP, Proteintech, Rosemont, IL, USA; Mouse anti α-SMA monoclonal antibody, 61001, Progen, Heidelberg, Germany), diluted 1:200 in 10% BSA in PBS. Secondary antibodies coupled with horseradish peroxidase (MP-7401/MP-7452) or alkaline phosphatase (MP-5401) and corresponding substrates (SK-4100) were used for detection (all purchased from Vector Laboratories, Inc., Burlingame, CA, USA). Unspecific isotype IgGs were used to control the specificity of the secondary antibodies. Sections were counterstained with hematoxylin to visualize nuclei.

All sections were eventually dehydrated and mounted with Pertex® (Medite, Burgdorf, Germany). Photographs were taken using a Leica DMRB microscope (Leica, Wetzlar, Germany) equipped with a Canon EOS 600D with Canon EOS Utility 2 software, version 2.14 (Canon, Tokyo, Japan).
