Panoramic 1000 slide scanner

Tissues were fixed, embedded, and processed as described for immunohistochemistry. The Comparative Pathology Laboratory (University of Iowa) completed histochemical staining with amylase‐pretreated Periodic acid‐Schiff (dPAS). High‐resolution images of the slides were acquired with the 20× objective at 0.24 μm/pixel resolution by the Panoramic 1000 slide scanner (3DHISTECH). Tissue sections were visualized using the 3DHISTECH CaseViewer software (3DHISTECH) to quantify histological features of all tissue specimens. To provide a standardized sampling for measurements, the right medial liver lobe was sectioned containing the gallbladder. A liver parenchyma area of at least 30 mm² was evaluated and all ducts within the liver section that had a discernable lumen were quantified. Quantitative (duct diameter and lumen diameter) and semi‐quantitative (mucus scoring) measures were performed in a manner blinded to the group to avoid bias (Meyerholz & Beck, 2018 (link)). To account for different sectioning planes, the minor diameter was measured for the duct and lumen diameters. We studied mucus using the dPAS stain (Meyerholz et al., 2018 (link)a). Ordinal scoring of mucus had 4 levels: 0–no dPAS staining present in duct, 1–dPAS staining present intracellularly or on the cell surface, 2–dPAS staining present in lumen, 3 – lumen obstructed with dPAS material, bile, or cellular material.

Microglial and astrocytic activation was assessed using the inflammation markers, Iba1 (1:1000, Wako Ltd., Japan) and GFAP (1:1000, Sigma, Germany) for the fresh-frozen spinal cord slices collected for the autoradiography studies. The immunohistochemistry procedures were performed as described before [22 (link), 23 (link)]. Images of the stained slices were taken with a Panoramic 1000 slide scanner (3DHISTECH).