Rotary microtome microm hm 340e

Mouse liver tissues were directly fixed in 3.7% paraformaldehyde (Merck) for 24 h and stored in 70% ethanol before paraffin embedding and 4.5 μm sectioning using the Rotary Microtome Microm HM 340E (Thermo Scientific). Hematoxilin (MilliporeSigma, 51275) and eosin (MilliporeSigma, E4382) (H&E) staining was then performed. Anti-cytokeratin 7 (CK7) staining was performed on liver sections blocked with normal antibody diluent (ImmunoLogic) and incubated with (1:8,000) rabbit anti-CK7 (Abcam, ab181598) overnight at 4°C. After incubation with BrightVision anti-IgG poly-horse radish peroxidase-conjugated secondary antibody (ImmunoLogic), Vector NovaRED peroxidase substrate (Brunschwig, Basel, Switzerland) was used to visualise horse radish peroxidase. Digital imaging of the section was done using an Olympus BX-51 microscope (Olympus), equipped with a 20× objective. Tile scans were made using the 5× objective of the Leica DM600B microscope, and necrotic areas were scored as percentage (surface) of the total liver section using ImageJ. CK7 positive stain was quantified by colour deconvolution using ImageJ FIJI.

For histological analysis of mouse colons, distal colons containing fecal content were isolated at day 12 p.i. and fixed for 3 hr at room temperature (RT) in freshly prepared Carnoy’s fixative [60% anhydrous methanol (Sigma-Aldrich), 30% chloroform (Sigma-Aldrich), 10% glacial acetic acid (Sigma-Aldrich)]. Fixed colons were transferred to fresh Carnoy’s fixative and fixed again overnight. Fixed samples were washed in anhydrous methanol for 2 hr followed by transfer to fresh methanol and storage at 4°C until further use. Using a Tissue-Tek VIP processor (SAKURA), samples were subjected to consecutive washes with 100% denatured ethanol (VWR), treatment with the clearing agent toluene (VWR), and paraffin embedment (Leica). Sections (3µm) were cut using a Rotary Microtome Microm HM 340E (Thermo Fisher Scientific). Paraffin-embedded sections were either stained with hematoxylin (Medite) plus eosin (VWR) (H&E), or Alcian Blue (Dako). Alcian Blue staining was performed using an Artisan Link Pro Special Staining System (Dako). Slides were scanned with an automated digital slide creation and viewing system (Philips IntelliSite Pathology Solution; 760001).