EHBDs were clarified and imaged in Murray’s clear (2:1 benzyl benzoate/benzyl alcohol) before analysis by confocal microscopy. Immunofluorescence (IF) and light microscopy imaging was performed using an Olympus microscope and DP71 camera (Olympus, Tokyo, selleckchem Japan). Images were processed with Olympus DP Controller/Manager software. Whole-mount

imaging was performed using one of two confocal microscopes as follows: (1) Zeiss LSM 510 on an Axiovert 200-M inverted microscope, operating with AIM 4.0 software or (2) Nikon A1R SI on a Nikon TI-E inverted microscope, operating with NIS Elements 4.0 software. 3D renderings of confocal images were computer-generated using either ZEN 2009 Light Edition or Imaris Version 7.5 software on an HP Z400 Workstation. EHBDs were snap-frozen in optimal cutting temperature (OCT) medium, sectioned using a cryostat,

fixed in ice-cold 3.7% formalin for 20 minutes, and blocked in 10% normal donkey serum. For CK staining, sections were incubated with undiluted rabbit anti-CK primary Ab or goat anti-mouse CK-19 Ab and a second primary Ab based on the protein of interest, as follows: goat anti-Pdx1 (ab47383, diluted at 1:50,000; Abcam, Cambridge, MA), goat anti-Sox17 selleck screening library (AF1924, diluted at 1:2,000; R&D Systems, Minneapolis, MN), and rabbit anti-mouse chromogranin A (CgA; ID #20085, diluted at 1:5,000; Immunostar, Hudson, WI). CK Ab incubations were performed for 1 hour at RT, whereas all other primary Ab incubations were performed overnight at 4oC. To detect specific signals, sections were then counterstained with species-specific Abs for 1 hour at RT. Periodic acid-Schiff (PAS) or Alcian blue stainings were performed in paraffin-embedded sections using established protocols.[15, 16] We injected 1.5 × 106 fluorescence-forming medchemexpress units of rhesus rotavirus (RRV) or 0.9% NaCl (saline) solution intraperitoneally (IP) into Balb/c mice within 24 hours of birth, as described previously.[17] Newborn mice were housed with their mother in a specific pathogen-free

vivarium room with a 12-hour dark/light cycle. Groups of mice were sacrificed at 3-7 days, and EBHDs were microdissected and snap-frozen in liquid nitrogen. All mice received appropriate care that is consistent with criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (Bethesda, MD). The institutional animal care and use committee of the Cincinnati Children’s Research Foundation approved all animal protocols. Surgical BDL was performed in Balb/c mice at 3 months of age. For the procedure, mice underwent general anesthesia, and after a small right subcostal incision, the CBD was identified, separated from the surrounding tissue, and ligated by deployment of a small titanium ligation clip (LigaClip-LT100; Ethicon Endo-Surgery, Inc., Cincinnati, OH) in the lower third of the CBD.