This protocol enables the investigator to quantify bile duct development in mouse models of liver disease. It also allow for evaluating the effects of genetic modifiers on bile duct development. The advantage of this technique is that it is a straightforward, sensitive and quantitative method for the direct assessment of bile duct development.
To begin, with the skin held taut by forceps, use small scissors to make a transverse incision approximately one inch below the rib cage of a euthanized mouse. Expose the entire ventral surface of the liver. Use small scissors to carefully cut through the ligaments connecting the liver to the other organs in the abdomen.
Then, cut through the common bile duct to detach the liver from the intestine. Hold on to the gallbladder and carefully remove the liver. Immediately place it in a 50 milliliter tube filled three-quarters with 4%paraformaldehyde.
To fix the liver tissue, incubate it in 4%PFA at four degrees Celsius for 48 hours. After a series of ethanol washes, wash the liver with clearing agent three times for 30 minutes each at room temperature. To begin embedding, wash a tissue cassette in a tissue mold three times for 30 minutes in paraffin wax preheated to 60 degrees Celsius.
Then, fill the tissue mold with paraffin wax to three-quarters height and keep it on a heating block at 60 degrees Celsius. Place the liver in the mold with the ventral side facing up. After carefully removing the mold from the heating block, place the top of the cassette on the mold.
Top off with hot liquid paraffin and allow it to cool to room temperature overnight. After removing the liver block from the mold, use a microtome to begin sectioning through the superficial dorsal side of the liver, making five micrometer sections. Check the superficial sections under a dissection microscope to ensure the sections are not sheared or folded.
To process slides for immunohistochemistry, select one slide per genotype to be analyzed and wash it for 15 minutes in xylene, 100%ethanol, 95%ethanol and finally, 70%ethanol. After washing the slide in the ionized water, immerse it in Tris-based high pH antigen retrieval solution. Then, heat it under pressure in a pressure cooker for three minutes at 10 pounds per square inch.
After letting the slide cool to room temperature, use a PAP pen to outline the sections on the slide. After washing with PBS Tween, block the tissue sections by adding 100 microliters of blocking solution per section and incubate covered at four degrees Celsius for one hour. Apply 100 microliters of the diluted antibody solution containing all three primary antibodies to each section and incubate them at four degrees Celsius overnight.
After washing the slides, add 100 microliters of the secondary antibody solution containing both secondary antibodies and incubate for one hour at room temperature. Lastly, apply mounting medium and a coverslip to the slides. Use a fluorescent microscope to take 20x images at 1x zoom of each section.
Ensure that every portal vein across the liver is imaged. Create a spreadsheet with the following columns:Animal/Sample number, Image number, number of portal veins and number of bile ducts. Identify and record the number of portal veins per image for each image.
Then, identify patent bile ducts in each image, by the presence of cholangiocytes surrounding a definable lumen. These structures should be distinct and separated by mesenchyme from other wide-spectrum cytokeratin-positive cells. One main challenge of this technique is in identifying patent bile ducts.
Determining what is a patent duct requires an analysis of the shape of the duct and the cells surrounding the lumen. Count each patent bile duct and place in the same column as the image number and repeat for each portal vein image. Finally, calculate the sum of all portal veins and all bile ducts in the liver sample and calculate the bile duct to portal vein ratio for the liver sample.
P30 mouse livers were sectioned and co-stained for CK8 and CK19 along with the vascular marker, alpha-SMA, to determine the BD to PV ratio. When all the portal veins in each liver lobe are imaged, portal veins were defined as alpha-SMA stained vessels that have adjacent wide-spectrum cytokeratin staining. The alpha-SMA stain structures without wide-spectrum cytokeratin were central veins which were excluded from the analysis.
Patent ducts have a clearly definable lumen that is surrounded by wide-spectrum cytokeratin-positive cholangioctyes and are usually separated from nearby ducts or cholangiocytes by mesenchyme. Wide-spectrum cytokeratin-positive cells that do not have a definable lumen, are not counted toward the total number of bile ducts. Liver section from a wild-type animal shows a portal vein associated with a fully patent duct along with several unincorporated cells.
Representative liver section from a JAG1 heterozygous animal shows no patent bile ducts are present around the three portal veins. All wide-spectrum cytokeratine-positive cells here are unincorporated and therefore, should not be counted. Analysis of the BD to PV ratio for three wild-type and three JAG1 heterozygous animals shows how the two genotypes can be readily distinguished based on bile duct counts.
It is important to assess all portal vessels for bile ducts. It's especially critical for determining bile duct density if there is phenotypic variability across the liver. This technique was used to identify a genetic suppressor of the biliary phenotype in a mouse model of Helgason.
Therefore, it may be useful in assessing treatment modalities of animal models of biliary disease.
