The authors acknowledge Johanna Hagens, Pauline Schuppert, Clara Philippi, PD Dr. med Christian Tomuschat, Svenja Warnke, PD Dr. Diana Lindner, Prof. Dr. Dirk Westermann, Miriam Tomczak, Nicole L&#252;der, Nadine Kurzawa, Dr. rer nat. Laia Pagerols Raluy, Birgit Appl, and Magdalena Trochimiuk for their contributions.&#160;Hans Christian Schmidt was financially supported by the Else Kr&#246;ner-Fresenius-Stiftung iPRIME Scholarship (2021_EKPK.10), UKE, Hamburg.

Following ethical approval (N045/2021), male and female C57BL/6 mice neonates were observed until 9 days old. The animals were born and provided for experimental purposes by the animal facility of the University Medical Center Hamburg-Eppendorf, Hamburg, Germany. The neonates were housed in a cage together with their parent animals. The environmental conditions were controlled in temperature (20-24 &#176;C), 12:12 h light-dark cycle, and relative humidity of 40%-70%.
1. Experimental prep...

<ol>
	<li>Liwinski, T., Schramm, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prim&#228;r+sklerosierende+Cholangitis.">Prim&#228;r sklerosierende Cholangitis.</a> Der Internist. 59 (6), 551-559 (2018).</li><li>Kobayashi, H., Stringer, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+atresia.">Biliary atresia.</a> Seminars in Neonatology. 8 (5), 383-391 (2003).</li><li>Patman, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biliary+tract:+Newly+identified+biliatresone+causes+biliary+atresia.">Biliary tract: Newly identified biliatresone causes biliary atresia.</a> Nature Reviews Gastroenterology &amp; Hepatology. 12 (7), 369 (2015).</li><li>Mack, C. L., Sokol, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unraveling+the+pathogenesis+and+etiology+of+biliary+atresia.">Unraveling the pathogenesis and etiology of biliary atresia.</a> Pediatric Research. 57 (5), 87-94 (2005).</li><li>Karjoo, S., Wells, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+neonatal+extrahepatic+cholangiocytes.">Isolation of neonatal extrahepatic cholangiocytes.</a> Journal of Visualized Experiments. (88), e51621 (2014).</li><li>Strazzabosco, M., Fabris, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy+of+normal+bile+ducts.">Functional anatomy of normal bile ducts.</a> The Anatomical Record: Advances in Integrative Anatomy and Evolutionary Biology. 291 (6), 653-660 (2008).</li><li>Nakanuma, Y., Hoso, M., Sanzen, T., Sasaki, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microstructure+and+development+of+the+normal+and+pathologic+biliary+tract+in+humans,+including+blood+supply.">Microstructure and development of the normal and pathologic biliary tract in humans, including blood supply.</a> Microscopy Research and Technique. 38 (6), 552-570 (1997).</li><li>Banales, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholangiocyte+pathobiology.">Cholangiocyte pathobiology.</a> Nature Reviews Gastroenterology &amp; Hepatology. 16 (5), 269-281 (2019).</li><li>de Buy Wenniger, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cholangiocyte+glycocalyx+stabilizes+the+'biliary+HCO3-+umbrella':+an+integrated+line+of+defense+against+toxic+bile+acids.">The cholangiocyte glycocalyx stabilizes the 'biliary HCO3- umbrella': an integrated line of defense against toxic bile acids.</a> Digestive Diseases. 33 (3), 397-407 (2015).</li><li>Pinto, C., Giordano, D. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high-purity+myenteric+plexus+from+adult+human+and+mouse+gastrointestinal+tract.">Isolation of high-purity myenteric plexus from adult human and mouse gastrointestinal tract.</a> Scientific Reports. 5 (1), 9226 (2015).</li><li>Ishii, M., Vroman, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+morphologic+characterization+of+bile+duct+epithelial+cells+from+normal+rat+liver.">Isolation and morphologic characterization of bile duct epithelial cells from normal rat liver.</a> Gastroenterology. 97 (5), 1236-1247 (1989).</li><li>Kumar, U., Jordan, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+biliary+epithelial+cells+from+the+biliary+tract+fraction+of+normal+rats.">Isolation and culture of biliary epithelial cells from the biliary tract fraction of normal rats.</a> Liver. 6 (6), 369-378 (1986).</li><li>Vroman, B., LaRusso, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+characterization+of+polarized+primary+cultures+of+rat+intrahepatic+bile+duct+epithelial+cells.">Development and characterization of polarized primary cultures of rat intrahepatic bile duct epithelial cells.</a> Laboratory Investigation. 74 (1), 303-313 (1996).</li><li>Paradis, K., Sharp, H. 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The authors declare no conflict of interest.

This article reported and discussed the creation and validation of a new surgical approach for removing the EBDS of euthanized neonatal mice. Microscopical and histological findings reveal that the approach quickly detects EBDs and dissects them near the duct&#39;s margins, even in neonatal mice. Only surgical instruments and a microscope with a 20x magnification are required for the described protocol. Furthermore, the approach allows the isolation of the entire EBDS. The technique is highly efficient, straightforward, ...

The genesis and progression of cholangiopathies such as biliary atresia, primary sclerosing cholangitis (PSC), and primary biliary cholangitis (PBC) are either unknown or incomplete1,2. The limited understanding of the origin and progression of those diseases leads to a paucity of therapy options3. The most difficult obstacle in studying neonatal bile duct disorders is gaining a molecular understanding of the pathophysiology. One of the essential keys to a better understanding of molecular pathology is the best possible observation of affected tissue. To avoid reduced comparability and discrepancies between research, such as observing potential viral etiology of biliary atresia4,&#160;the need for the best possible preparation and sharing of the performed dissection techniques arise. A pure preparation of the target tissue is necessary for later microscopical investigations or breeding cell- and 3D-organoid cultures. However, in murine neonatal disorders, tissue samples are rare and only occur in a small amount due to the very small size. Regarding bile duct disorders, difficulties in a clean preparation of bile ducts in murine neonates have been described5. Due to the neonatal stage of development, tissue differentiation is not overly advanced, which complicates preparation and increases the difficulty compared to the preparation of adult samples. Therefore, the operating workgroup investigated a novel strategy for preparing the EBDS in a neonatal mouse model. In the present study, the technique allows an efficientdissection of each sample.
The bile duct system is intraperitoneally placed in the right upper abdomen, arising from the liver. The gall bladder is located underneath the visceral surface of the liver&#39;s right lobe. The bile duct, together with the portal vein and hepatic artery, is embedded in the hepatoduodenal ligament. It joins the liver and the duodenum directly and drains bile fluid into the duodenum6. Anatomically, the bile duct is divided into the right and left hepatic ducts, the common hepatic duct, the cystic duct, and the Ductus choledochus, which is formed by the confluence of the cystic duct and the common hepatic duct7. This one eventually empties bile fluid and saliva from the pancreatic duct into the duodenum via the Ampulla of Vater.
Cholangiocytes line the bile duct intra- and extrahepatically, dwelling in a complicated anatomic niche where they assist in bile production and homeostasis8. Bile fluid passes these specialized epithelial cells in high concentrations daily. In particular, the HCO3- umbrella maintenance is very important for protecting against bile acid toxicity9. Cholangiocytes are the first line of defense in the hepatobiliary system against, for example, luminal microorganisms10. The cholangiocytes&#39; defense efficacy against toxic assaults may be weakened by genetic predisposition. A toxic overload causes damage and destruction and can therefore lead to cholangiopathies. Furthermore, the developing bile duct is not completely capable of all self-protective mechanisms, leading to a higher susceptibility to environmental toxins in neonatal bile ducts11.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Propanol</td>
			<td>CHEMSOLUTE</td>
			<td>11365000</td>
			<td>used as a dehydrating agent</td>
		</tr>
		<tr>
			<td>30 G canula</td>
			<td>B Braun/Sterican, Melsungen Germany</td>
			<td>4656300</td>
			<td>canula for hydration of the sample</td>
		</tr>
		<tr>
			<td>Air vent</td>
			<td>C + P M&#246;belsysteme GmbH &#38; Co. KG, Breidenbach, Germany</td>
			<td>Tec-Ononmic AZ 1200</td>
			<td>the use of an air vent helps to avoid inhalation of formalin-containing fixatives</td>
		</tr>
		<tr>
			<td>Aqua ad injectabilia Braun</td>
			<td>B Braun, Melsungen, Germany</td>
			<td>2351744</td>
			<td>saline; Container: Mini-Plasco connect, 20 x 10 mL, sterile</td>
		</tr>
		<tr>
			<td>Bigger microsurgical Forceps</td>
			<td>DIADUST von Aesculap, Trossingen Deutschland</td>
			<td>FD253R</td>
			<td>straight, 180 mm (7&#34;), platform tip, round handle, width: 0,800 mm, diamond dust coated, non-sterile, reusable optional tool for observation and every step of preparation except very final preparation; Dividing skin of the peritoneum</td>
		</tr>
		<tr>
			<td>Camera &#8220;SmartCAM 5&#8221;&#160;</td>
			<td>Basler and Vision Engineering, Send, United Kingdom</td>
			<td>EVC131A</td>
			<td>optional Lynx Exo camera modul: sensortype: CMOS, resolution 2560 x 1920 pixels, sensor size: 1/2&#34;; Used for videoproduction and technical evaluation</td>
		</tr>
		<tr>
			<td>Dehydration machine/Citadel 2000 Tissue Processor</td>
			<td>Fisher Scientific GmbH, Schwerte, Germany</td>
			<td>12612613</td>
			<td>used for automatic dehydration, short program (approx. 4.8 h)</td>
		</tr>
		<tr>
			<td>Dehydration sponge&#160;</td>
			<td>Carl Roth, Karlsruhe, Germany</td>
			<td>TT56.1</td>
			<td>sponge for final dissection step, other sponges/foam pads with a minimum pore size of 60 pores per inch are also suitable, the use of&#160; two foam pads per embedding cassette is recomended to cover the sample from below and above to prevent sliding through the perforation of the embedding cassettes</td>
		</tr>
		<tr>
			<td>Dulbecco&#180;s Phosphat Buffered Saline (PBS)</td>
			<td>Gibco</td>
			<td>14190-144</td>
			<td>Doesn&#180;t contain Calzium or Magnesium, 500 mL</td>
		</tr>
		<tr>
			<td>Embedding cassettes</td>
			<td>Engelbrecht GmbH, Ederm&#252;nde, Germany</td>
			<td>17990</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>MEDITE Medical GmbH, Burgdorf, Germany</td>
			<td>41-6660-00</td>
			<td>staining solution, ready to use</td>
		</tr>
		<tr>
			<td>Fine Scissors CeramaCut</td>
			<td>FST, Heidelberg Germany</td>
			<td>14959-09</td>
			<td>Tips: Sharp-Sharp, Alloy / Material: Ceramic Coated Stainless Steel, Serrated:, Yes; Feature: CeramaCut, Tip Shape: Straight, Cutting Edge: 22 mm, Length: 9 cm; Skin incision, incision of the peritoneal window</td>
		</tr>
		<tr>
			<td>Graefe Forceps</td>
			<td>FST, Heidelberg Germany</td>
			<td>11051-10</td>
			<td>Length: 10 cm, Tip Shape: curved, serrated, Tip width: 0.8 mm, Tip Dimensions: 0.8 x 0.7 mm, Alloy /Material: Stainless Steel</td>
		</tr>
		<tr>
			<td>Hematoxylin</td>
			<td>MEDITE Medical GmbH, Burgdorf, Germany</td>
			<td>41-5130-00</td>
			<td>staining solution, ready to use</td>
		</tr>
		<tr>
			<td>Highresolotion microscope</td>
			<td>Vision Engineering, Send United Kingdom</td>
			<td>EVO503&#160;</td>
			<td>Capable of enlargement up to 60x magnification, only 6x to 20x magnification were used&#160;</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus Optical CO, Ltd., Hamburg, Germany</td>
			<td>BX60F5</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glases</td>
			<td>Marienfeld, Lauda-K&#246;nigshofen, Germany</td>
			<td>101244</td>
			<td>60 mm broad, made of SCHOTT D 263 glass</td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>R. Langenbrinck GmbH, Emmendingen, Germany</td>
			<td>03-0060</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica, Nu&#223;loch, Germany</td>
			<td>SM2010R</td>
			<td>Tool for sectioning (2 &#181;m-slices)&#160;</td>
		</tr>
		<tr>
			<td>Omnifix-F 1 mL syringe</td>
			<td>B Braun, Melsungen, Germany</td>
			<td>9161406V</td>
			<td>syringe without canula</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sakura Finetec, Torrance, USA</td>
			<td>4511</td>
			<td>Tissue-Tek Paraffin Wax Tek III, without DMSO</td>
		</tr>
		<tr>
			<td>Paraffin embedding machine</td>
			<td>MEDITE Medical GmbH, Burgdorf, Germany</td>
			<td>TES 99</td>
			<td>The embedding machine used in this study contained the following three individual modules: TES 99.420, TES 99.250, TES 99.600. The sample should be embedded in Paraffin directly after the dehydration, no interim storage in a fridge should be performed due to possible shrinking and moisture in the fridge</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Morphisto</td>
			<td>1176201000</td>
			<td>Prepare 1 mL Aliquots in 2 mL Eppendorf conical Tubes for liver samples and 0.5 mL Aliquots in 1 mL Eppendorf conical Tubes for extrahepatic bile duct samples, 4% in PBS ph 7.4&#160;</td>
		</tr>
		<tr>
			<td>Small Microsurgical Forceps&#160;</td>
			<td>EPM (Erich Pfitzer Medizintechnik), B&#252;tthard, Bayern, Germany</td>
			<td>(00)165</td>
			<td>Round handle, straight, 0.3 mm tip, tool for observation and every step of preparation, especially useful in final preparation</td>
		</tr>
		<tr>
			<td>Stainless Steel Ruler</td>
			<td>Agntho&#39;s AB, Liding&#246;, Sweden</td>
			<td>30085-15</td>
			<td>150mm With Metric &#38; Inch Graduations</td>
		</tr>
		<tr>
			<td>Surgical Scissors &#8211; Sharp-blunt for decapitation</td>
			<td>FST, Heidelberg Germany</td>
			<td>14001-14</td>
			<td>Device for decapitation</td>
		</tr>
		<tr>
			<td>Warming cabinet</td>
			<td>Haraeus, Hanau, Germany</td>
			<td>T 6060</td>
			<td>the sliced samples should be kept in the warming cabinet to ensure the attachement of the sample on the microscope slides</td>
		</tr>
	</tbody>
</table>

extrahepatic bile duct and gall bladder dissection in nine-day-old mouse neonates

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation of the extrahepatic bile duct (EBD) in mouse neonates without damaging the bile duct during preparation. Because of its exceptionally close preparation compared to the cholangiocytes cell line and harvesting of the entire extrahepatic bile duct system (EBDS), the described approach is extremely useful in researching animal models of newborn bile duct disorders, such as biliary atresia. After euthanasia, the peritoneal cavity was accessed, and the bile duct system, duodenum, and liver were extracted with the unique En-bloc-Resection (EbR). The extracted sample is placed on a foam mat, and the EBD is dissected from contaminating cells atraumatically without necessary touch. The dissection of the entire EBDS is a significant advantage of this method. Caution must be taken due to the small size and amount of bile duct tissue. Using the described technique, there is no damage to the cholangiocytes. Further, the purity of the technique is reproducible (n = 10). Therefore, optimally comparable samples can be harvested. Furthermore, no bile duct tissue is harmed, because any contact with the bile duct system can be avoided during preparation, leaving the bile fluid inside the gall bladder. Most importantly, while performing the final gall bladder and bile duct dissection, atraumatic microinstruments were used only slightly lateral of the bile duct without squeezing it. This is the key to a clean and intact sample, and essential for further histological investigation or the isolation of cholangiocytes. To summarize, the described innovative dissection technique enables especially inexperienced operators with the necessary equipment to isolate the EBDS as cleanly as possible.

Figure 1A shows the EBDS of a murine neonate, which was dissected with the described technique. Microscopically, no further hepatic tissue is visible. The hepatic tissue has been removed during the final isolation steps of the protocol and could easily be distinguished from bile duct tissue regarding color and consistency. Figure 1B displays the isolated sample compared to a millimeter scale. The EBD&#39;s length (measured from gall bladder to duodenal papilla) ...

Watch this Scientific Journal Video about Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates at JoVE.com

Extrahepatic Bile Duct and Gall Bladder Dissection in Nine-Day-Old Mouse Neonates

For the observation of murine neonatal bile duct disorders, an intact bile duct and efficient preparation are required. Therefore, a new approach for isolating the entire extrahepatic bile duct system in murine neonates was successfully developed while maintaining the integrity of the bile duct.

The dissection of murine neonatal bile ducts has been described as difficult. The main aim of the described standard operating procedure is the isolation ...

Many reports assessing the extrahepatic biliary system reported that they obtained the samples but not how they obtained them, raising issues regarding comparability. The main advantage of this technique is that the entire extrahepatic biliary system can be dissected and contaminating cells can be removed atraumatically. Our dissection protocol can be generally applied to experimental approaches working on murine neonatal bile duct disorders, aiming for the dissection of the extrahepatic biliary system.Some features of the protocol, for example, leaving the stomach attached to the duodenum, might be useful in research of duodenal atresia to allow correct orientation by the partially-digested milk expressed by the stomach. At the beginning, the technique may necessitate some time for training. Practicing and following the step-by-step approach will increase speed and outcome.To begin, place the disinfected and autoclaved instruments on a sterile surface next to the operation table. After making a cut in the peritoneum, expand the cut to the location of the decapitation, following the left frontal axillary line. Remove the skin from the left to the right side with atraumatic forceps.Under a microscope, hold the peritoneum surrounding the spleen. Gently lift it up until the peritoneum resembles a tent-like structure, and cut a one-millimeter hole in the center. Wait for the peritoneal tent to be filled with air.Cut off the peritoneum in a window framed by the lower ribs, both lateral abdominal regions, and the lower bladder area. Ensure full access to the liver, bile duct system, stomach, small intestines, and colon. Examine the gallbladder and bile ducts by carefully pulling the xiphoid process in a cranial ventral position.Avoid tearing of the falciform ligament that can lead to tearing of the gallbladder from the bile duct system. Then, release the pull of the xiphoid process. Gently pull down the duodenum to free the bile duct system.Then, perform the lower En-bloc mobilization by identifying the duodenal papilla, which connects the bile duct system to the duodenum. Cut through the duodenum about two centimeters to the right lateral side of the papilla. Then, cut through the pyloric area.Ensure that the contents of the stomach are present in the pyloric area between the cutting location and the duodenal papilla. To perform the upper En-bloc mobilization, gently pull the xiphoid process and get access to the falciform ligament. Make a one-centimeter cut through the falciform ligament between the gallbladder and close to the xiphoid process.Cut through the connecting structures between the liver and the thorax, such as the esophagus, inferior vena cava, thoracic aorta, and all ligaments that surround the bare area of the liver. Also, cut all dorsally-remaining tissue connections. Under 20 times microscopic magnification, place the En-bloc sample on a foam pad.Assemble the sample by reorganizing it in the correct anatomical position. Using gentle movement, flatten the oral and aboral part of the duodenum. Using atraumatic forceps, start the flattening movement at the duodenal papilla and continue to the cutting edges.Then, smooth out the white pulpy contents of the stomach in the oral part of the duodenum. Identify the oral and the aboral part of the duodenum to rule out probable bile duct rotation. Cut away large remnants of hepatic tissue before dissecting the bile duct.After a few scraping movements, transfer the sample to a cleaner position on the foam mat. Use the less-squeezed liver tissue in the background to optimize the view for differentiation between EBDS and unwanted cells. Process the hepatoduodenal ligament until the isolated EBDS remains.Observe the hepatic artery and portal vein arising as white and very delicate filament, approximately three to five millimeters orally off the duodenal papilla. Carefully, remove this delicate filament using a gentle pull to the left lateral side. Ensure that the scraping movements start from the bile duct system and lead to the liver boundaries.Using this protocol, EBDS was dissected from nine-day-old murine neonates. The length of the isolated EBDS from gallbladder to duodenal papilla was less than 10 millimeters, and the diameter of the delicate ductus choledochus varied from 0.05 to 0.2 millimeters. Hematoxylin eosin staining was performed on the longitudinal section of the EBDS with an open lumen.The cholangiocytes were identified surrounding the lumen as a monolayer and were dyed darker. Researchers should share and standardize operating techniques such as dissection and injection protocols to improve comparability and rapid usability of their studies and results. Preceding dissection will be advantageous for histological and single-cell approaches, such as the isolation of intra and extraperi-cholangiocytes, because it results in the reduction of cell culture contamination.

extrahepatic-bile-duct-gall-bladder-dissection-nine-day-old-mouse

Research

JoVE Journal

Medicine

1.7K visualizações.  University Medical Center Hamburg-Eppendorf. Muitos relatórios que avaliaram o sistema biliar extra-hepático relataram que obtiveram as amostras, mas não como as obtiveram, levantando questões relativas à comparabilidade. A principal vantagem desta técnica é que todo o sistema biliar extra-hepático pode ser dissecado e as células contaminantes podem ser removidas atraumaticamente. Nosso protocolo de dissecção pode ser geralmente aplicado a abordagens experimentais que trabalham com distúrbios do ducto biliar neonatal murino,...