Scn400

Liver fibrosis was assessed by Sirius red staining to quantify collagen deposition in liver sections [14 (link)]. Following Sirius red staining, slides were scanned by a digital scanner (SCN400; Leica Microsystems, Buffalo Grove, IL) and quantified by the Image-Pro Premier 9.1 (Media Cybernetics, Inc., Rockville, MD). In addition, immunofluorescence staining was performed for Col1a1 co-stained with CK-19 and desmin in frozen liver sections (8 μm). Immunofluorescent staining was visualized using Leica TCS SP5 X system (Leica Microsystems Inc.) and Olympus F300 from Texas A&M Integrated Microscopy Imaging Laboratory. We also measured the expression of Col1a1, Fn1 and TGF-β1 in isolated cholangiocytes and total liver samples by qPCR.

A tibia was extracted from another porcine lower leg, and the soft tissue was removed, leaving the periosteum intact. The tibia was divided into three segments and prepared as follows: the periosteum was left in the proximal 1/3 segment; the periosteum was peeled away from the tibia in the middle 1/3 segment; or the periosteum was peeled away from the tibia, and the outer cortex was scraped in the distal 1/3 segment.
After preparation, the samples were placed in a small container filled with gelatin gel, and MRI was performed using the same sequence as in experiment 1. The same radiologists who evaluated the images of the 1st experiment analyzed the images of prepared tissue with a consensus, but without awareness of the tissue preparation. They assessed the signal intensity around the outer surface of the tibia and graded it in the same manner as in the 1st experiment.
After the MRI scan, the three tibial segments were fixed using 10% buffered formalin, decalcified in Cal Ex II (Fisher Scientific) for 48 hours, and prepared for histologic analysis, as described in experiment 1. The histological sections were subjected to hematoxylin and eosin staining. The stained slides were digitally imaged at 340 magnification (model SCN400; Leica Microsystems) and examined to determine if the tissue preparation was successful.

Liver histology was analyzed using a previously described protocol.²⁷ (link) Liver tissues were immediately fixed in 10% formalin buffer after removal from the animals. The tissue samples were embedded in paraffin wax after alcohol dehydration process. By using microtome, 5 µm sections of liver tissue were taken and were stained by haematoxylin and eosin stain. The processed tissue sections were then visualized and captured using a Leica Scanner, SCN400 and Slide Path Gateway LAN software for analysis (Leica Microsystems CMS, Wetzlar, Germany).