title: Vital staining of blood vessels and bile ducts with carboxyfluorescein diacetate succinimidyl ester: a novel tool for isolation of cholangiocytes.
abstract: Current methods for visualization of the blood vasculature, biliary tree and for isolation of vital cholangiocytes are afflicted with a plethora of technical difficulties, especially in mice. In this project, we propose a novel, reliable and straightforward alternative technique for histological demonstration of blood- and biliary systems and derivation of vital cholangiocytes.
  
  
  
  Intravital retrograde perfusion of bile ducts was performed in twenty wild type mice. Liver and gallbladder were exposed by median laparotomy. Using a venous catheter, the gallbladder was cannulated, a few millimeters of the liver edge were cropped to allow free outflow of the perfusate, and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) solution was retrogradely infused. Thereafter, formaldehyde solution was either injected through the same catheter, or the liver was immediately dissociated into a single-cell suspension for FACS-analysis. Intravital perfusion of the vascular system was performed in ten Lewis rats by direct intra-arterial injection of CFDA-SE into the abdominal aorta. The specificity and sensitivity of CFDA-SE labeling was controlled using Indian ink or cytokeratin 19 immunohistochemistry respectively.
  
  
  
  Upon histomorphological analysis of cryo- and paraffin sections, strong fluorescence was noted in large and small bile ducts throughout the entire liver and in the vascular system after infusion of the CFDA-SE solution. In preliminary FACS-experiments, we succeeded in separating cholangiocytes based on combined CFDA-SE-staining and cell size.
  
  
  
  Visualization of liver architecture and the isolation of cholangiocytes is feasible using a fast and cost-effective method of retrograde perfusion and vital fluorescent labeling of mouse bile duct epithelium and vascular endothelium with CFDA-SE.
authors: Department of Gastroenterology and Hepatology, University Hospital of Cologne, Cologne, Germany. galyna.pryymachuk@uk-koeln.de
authors: Galyna
authors: G
authors: Pryymachuk
authors: Elias
authors: E
authors: Polykandriotis
authors: Stephanie
authors: S
authors: Schievenbusch
authors: Andreas
authors: A
authors: Arkudas
authors: Dirk
authors: D
authors: Nierhoff
authors: Harald-Morten
authors: HM
authors: Curth
authors: Margarete
authors: M
authors: Odenthal
authors: Raymund E
authors: RE
authors: Horch
authors: Wolfram F
authors: WF
authors: Neiss
authors: Tobias
authors: T
authors: Goeser
authors: Hans-Michael
authors: HM
authors: Steffen
authors: Ulrich
authors: U
authors: Toex
mesh_terms: D000818:Animals; D001652:Bile Ducts / Q000098:blood supply* / Q000473:pathology; D001653:Bile Ducts, Intrahepatic / Q000166:cytology; D001808:Blood Vessels / Q000473:pathology*; D004727:Endothelium / Q000473:pathology; D005260:Female; D005434:Flow Cytometry; D005452:Fluoresceins / Q000737:chemistry*; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes / Q000737:chemistry; D007150:Immunohistochemistry; D008297:Male; D051379:Mice; D010477:Perfusion; D051381:Rats; D015203:Reproducibility of Results; D013194:Staining and Labeling; D013388:Succinimides / Q000737:chemistry*; D014018:Tissue Distribution
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013388:Succinimides