Aperio scanscope cs2

Liver tissues obtained on day 6 post-infection were fixed in 10% buffered formalin and 4 μM paraffin-embedded sections were stained with hematoxylin and eosin. The specimen slides were scanned at 20 × magnification on Aperio CS2 Scanscope® (Leica Biosystems Inc.) followed by observer-blind histopathological analysis.

Immunohistochemistry was performed using the ABC-HRP protocol. All heat-induced epitope retrieval was performed with citrate buffer (pH = 6.0) except for CD4 staining, which used Tris-EDTA Buffer (pH = 9.0) and a methyl green counter stain. Please refer to Table A1 for a list of all antibodies used for IHC. All images were uploaded using Aperio CS2 ScanScope (Leica, Wetzlar, Germany).

Full lengths of the colons were prepared for fixation by rolling from distal to proximal as a “Swiss roll” with the mucosa facing inside, a previously described method to examine the full length of tissue in sections (Whittem et al., 2010 (link)). The rolled tissue was fixed in place with 4% formaldehyde in phosphate buffered saline for 48 h. Tissues were then transferred to 70% ethanol for 24 h and taken through steps of dehydration and embedded in paraffin. Sections (4 µm thick) were cut using a microtome, and hematoxylin and eosin staining was performed to visualize tissue morphology using standard histological methods, also previously described (Morohaku et al., 2013 (link); 2014a (link)). Slides were studied under a light microscope (DM1000-LED, Leica), and images were acquired using a HD camera (ICC50HD, Leica); full slides were also scanned at ×40 magnification using the Aperio CS2 Scanscope (Leica). Image analysis was performed to quantify proximal distance of ulcerated tissue showing epithelial loss. In brief, lengthwise measurements of colonic epithelial pathology (from distal to proximal) and total colon length were made from scanned histopathology images after calibration using Adobe Illustrator. These values were used to generate a ratio of affected to total colon length for comparison between the groups.

Immunofluorescence imaging was carried out using the LEICA fluorescent microscope (LEICA AF6000 Modular System) and Keyence BZX800. Slides stained for fibrosis were imaged with a Leica Aperio ScanScope® CS2.