Publication: Vital staining of blood vessels and bile ducts
with carboxyfluorescein diacetate succinimidyl
ester: a novel tool for isolation of cholangiocytes
Authors
Pryymachuk, Galyna ; Polykandriotis, Elias ; Schievenbusch, Stephanie ; Arkudas, Andreas ; Nierhoff, Dirk ; Curth, Harald-Morten ; Odenthal, Margarete ; Horch, Raymund E. ; Neiss, Wolfram F. ; Goeser, Tobias ; Steffen, Hans Michael ; Toex, Ulrich
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Publisher
F. Hernández y Juan F. Madrid. Universidad de Murcia. Departamento de Biología Celular e Histología
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DOI
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info:eu-repo/semantics/article
Description
Abstract
Background and aim: 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.
Methods: 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.
Results: Upon histomorphological analysis of cryoand
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.
Conclusions: 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.
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Citation
Histology and Histopathology, vol. 28, nº 8 (2013)
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