Bx51tf microscope

Formalin-fixed tissue samples were embedded in paraffin and sectioned at 5 μm. Sections were stained with hematoxylin and eosin for necrotic area measurements. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) assay was used to determine DNA strand breakage via the In Situ Cell Death Detection Kit (Roche Diagnostics, Indianapolis, IN). Additional sections for immunohistochemistry underwent a hydration process followed by a boiling citrate buffer (pH 6.0) antigen retrieval process, followed by 2 x 5 min TBS washes, and 3% hydrogen peroxide to block peroxidases. Liver sections were washed again, then blocked in 5% goat serum for 1h before being incubated with primary antibodies overnight at 4°C. Primary antibodies included anti-rabbit Cyp2e1 (1:100, Abcam, ab28146) and anti-mouse PCNA (1:500, Santa Cruz Biotechnology, sc-56). Liver sections were incubated for 30 min with HRP SignalStain Boost Detection reagent (CST, # 8114) or 1h incubation with m-IgG Fc BP-HRP (sc-529765) with SignalStain Dab Chromogen concentrate for visualization (CST, #8059). Images were acquired using an Olympus BX51TF microscope with an Olympus DP74 camera.

Fixed samples were cut into distal and proximal halves, embedded in paraffin, and sectioned at 5 µm thicknesses. Sections from the sample center were stained with Movat’s pentachrome to identify fibrin and collagen and with picrosirius red to identify different thickness collagen fibers. Additional sections were immuno-stained for α-smooth muscle actin (αSMA) to identify myofibroblasts, CD68 to identify macrophages, and Ly6b to identify neutrophils.
We acquired all histological images with an Olympus BX51TF microscope and an Olympus DP70 camera using bright-field imaging, except for the picrosirius red stained sections, which were acquired using polarizing optics and dark-field imaging. Both bright-field and dark-field images were taken at 10× and 20× magnification. We performed quantitative measurements using ImageJ (NIH) and an in-house Matlab image processing software.

Hematoxylin & eosin stained sections were prepared in the Histology Core of our Digestive Disease Research Center. Images were obtained by Olympus BX51TF microscope (Olympus Corporation, Tokyo, Japan). Apical sodium-dependent bile acid transporter protein (ASBT) expression in the terminal ileum was assessed by immunofluorescence. Sections were incubated with anti-ASBT antibody (Santa Cruz Biotechnology, Dallas, TX) and positively stained area was measured using Image-Pro® Plus software (Media Cybernetics, Bethesda, MD).