The overall goal of this procedure is to employ whole-mount immunohistochemistry staining for the visualization of the nerve distribution within the biliary tract of Suncus murinus. This method can be used to analyze the nerve distribution of the visceral organs of many species, particularly irregular or small organs that are difficult to assess by standard methods. The main advantages of this technique are that the operation is simple.
It does not require complicated procedures and it has a high success rate. Demonstrating the procedure with me will be Yidan Dai, a graduate student, and Shuang-Qin Yi, a research fellow, both from my laboratory. Begin by flushing the animal with individual PBS and 4%paraformaldehyde perfusions in a fume cupboard.
Then inject two to three milliliters of white neoprene latex through the perfusion catheter into the abdominal aorta to label the blood vessels. And when all of the tissues of interest have been labeled, extract the abdominal organs and their related nerves and vessels. Then inject approximately 0.1 milliliters of blue neoprene latex into the gallbladder to label the biliary system and post-fix the tissues in 4%paraformaldehyde overnight at four degrees Celsius for whole-mount immunostaining.
The next morning, transfer the sample into a glass vial for four one-hour room temperature washes in distilled water on a nutator. After the last wash, incubate the tissues in freshly-prepared 1%orthoperiodic acid for 20 minutes at room temperature to prevent any intrinsic peroxidase reactions. Next, wash the sample in distilled water for 10 minutes at room temperature, followed by incubation in freshly-prepared 0.004%papillon for two hours in 37 degrees Celsius constant temperature water bath with gentle rocking.
At the end of the incubation, wash the specimen with distilled water for 50 to 60 minutes. Then store the tissues in 4%paraformaldehyde at four degrees Celsius overnight. The next morning, wash the sample in distilled water as just demonstrated followed by another two-hour incubation in freshly-prepared papillon.
Wash the sample for another 50 to 60 minutes in distilled water, followed by overnight fixation in 4%paraformaldehyde. On the third morning, wash the tissues four times in distilled water, followed by immersion in three sequential ascending 30-minute sucrose incubations. Then freeze the sample at minus 20 degrees Celsius for 30 to 60 minutes, followed by complete thawing at room temperature.
After the third freeze-thaw cycle, transfer the sample into 2%Triton X-100 for a four degree Celsius overnight incubation. The next morning, transfer the sample into a container of the primary antibody of interest for gentle rocking on the nutator at four degrees Celsius for three to six days according to the size of the specimen. After the appropriate labeling period, remove the unbound primary antibody with four one-hour washes in PBS at room temperature and label the specimen with the appropriate secondary antibody for three days with gentle rocking at four degrees Celsius.
At the end of the secondary antibody labeling period, wash the specimen four times in PBS. Then incubate the tissues in 10 microliters of 30%hydrogen peroxide in 100 milliliters of freshly-prepared 0.002%DAB coloration solution at four degrees Celsius for 16 to 72 hours with gentle rocking. When the optimal staining intensity has been reached, transfer the specimen into 0.04%sodium azide in PBS to stop the coloration reaction.
Then carefully immerse the sample in a 10 centimeter Petri dish containing fresh PBS and capture whole-mount images of the tissues using a camera mounted on a stereo microscope. In addition to visualization of the innervation of the extrahepatic biliary tract, labeling with antibody against neurofilament positive nerve fibers also allows the unambiguous identification of the running and distribution density of the vagus nerve along the esophagus and into the stomach. Labeling the blood vessels of the gallbladder with white neoprene latex reveals thin nervous fibrosis in high contrast to the surrounding tissues with a higher density of innervation observed in the neck of the gallbladder than in the fundus.
In the upper bile duct, two types of nerve bundles are labeled, the fine nerve bundles which form an irregular and dense network of nerves and run adhesively residing on and in the biliary tract and the thicker neural bundles that are distributed parallel to the surface of the biliary tract. Labeling of the common bile duct with blue neoprene latex and the vessels with white neoprene latex allows visualization of the thin nervous fibers at the end of the common bile duct and the duodenal papilla area. Before attempting this procedure, take care to play out the timeline for each of the experiments as the entire process requires two to three weeks to be completed.
To avoid damaging the specimen when changing the solutions, always pour out the solutions instead of removing the specimen with forceps. After watching this video, you should be able to apply this technique to the labeling of a range of nerves within various visceral organs.
