Colonic tissue histology and histochemistry

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).