Imagescope 12

The CGRP immunoreactivity of α-CGRP receptor components (CALCRL) was analyzed by immunohistochemistry in paraffin-embedded liver sections (4 μm thick). A digital scanner (Aperio Scanscope CS System; Aperio Digital Pathology, Leica Biosystems, Milan, Italy) was used to scan the slides; the analysis was performed by an ImageScope 12.3.3 (Leica Biosystems) for each experimental group (n = 7 animals per groups; five different areas were analyzed from each group of mice). We performed qPCR for CALCA (gene for α-CGRP) in isolated cholangiocytes from WT mice fed the BAC or TC diet for 1 and 2 wk.

Biliary senescence was evaluated by staining for SA-β-galactosidase (SA-β-GAL) using commercially available kits (MilliporeSigma, Billerica, MA, USA) in frozen liver sections (10 µm thick); a semiquantitative analysis of the positive staining was performed for each group of mice (n = 3 animals per group; we analyzed five different areas from each group of mice). The slides were scanned by a digital scanner (Aperio Scanscope CS System; Aperio Digital Pathology, Leica Biosystems) and evaluated with ImageScope 12.3.3 (Leica Biosystems). Biliary senescence was evaluated by immunofluorescence for p16 in frozen liver sections (6 µm thick, co-stained with CK19) from the selected experimental groups. For all immunoreactions, negative controls (the primary antibody was replaced with bovine-serum-albumin) were performed; pre-immune serum was also added. Immunofluorescent staining was analyzed with an SP8 confocal microscope platform from Leica Microsystems (Leica Microsystems Inc., Buffalo Grove, IL, USA). To further support the immunohistochemical analysis, we measured the mRNA expression of p21 and p16 in isolated cholangiocytes from the experimental groups of mice by qPCR.

IBDM was measured by semiquantitative immunohistochemistry in paraffin-embedded liver sections (4 µm, thick) as the area occupied by % CK19-positive bile ducts over total liver area. Negative controls with the omission of the primary antibody (replaced with normal serum from the same species) were included for all samples. The slides were scanned by digital scanner (Aperio Scanscope CS System; Aperio Digital Pathology, Leica Biosystems) and a semiquantitative analysis of the positive staining was performed for each experimental group (n = 3 animals per group) and processed by ImageScope 12.3.3 (Leica Biosystems).

Stained slides were scanned in ScanScope™ system (Aperio, Vista, California, USA) with a resolution of 0.23 μm per pixel, and the generated digital slides were viewed in Imagescope 12 (Leica Biosystems, Nussloch, Germany). Quantification of SR and IHC stainings were carried out using the color deconvolution algorithm version 9 (Aperio), and the red, green, and blue (RGB) values for SR or DAB were obtained as described elsewhere (Ruifrok and Johnston 2001; Osman et al. 2013; Rosa et al. 2013). The obtained percentages of positive pixels were compared between different treatment groups.

Mouse hearts were excised and placed in 4% paraformaldehyde (7.4 pH) for 24 hours, rinsed with PBS and dehydrated with ethanol. After dehydration, mouse hearts where embedded in paraffin wax and sectioned (5 μm) for histology, conducted as previously described [39 (link),43 ].
To assess inflammatory cell infiltration, longitudinal heart sections were rehydrated and incubated with 0.5% Toluidine Blue in 0.5 N HCL for 3 days at room temperature, followed with a 10 minute wash in 0.7 N HCL, and were counterstained with Eosin (1:4). Mast cells were counted using an Axioskop transmitted light microscope (Carl Zeiss).
Cardiac remodeling was evaluated by collagen deposition and was determined using Sirius Red/Fast Green staining. Two series of sections were obtained from each heart, with the second series of histology samples sectioned approximately 50 μm deeper into the embedded mouse hearts, to provide a measure of collagen deposition throughout the heart. Heart sections were rehydrated and incubated with Picro Sirius Red supplemented with Fast Green. Sections were scanned using a ScanScope CS2 slide scanner, analyzed with ImageScope 12 (Leica Biosystems), and collagen deposition was determined as a percentage of the total tissue area, i.e. the relative area of collagen (Pico Sirius Red staining) to the total area of the tissue.