A New Clarification Method to Visualize Biliary Degeneration During Liver Metamorphosis in Sea Lamprey (Petromyzon marinus)

The overall goal of this procedure is to clarify the entire liver and the junction with the intestine for global, structural and cellular analyses of biliary degeneration. In metamorphic C lamprey. This is accomplished by first fixing the tissues in the hydrogel monomer solution.

Next, the hydrogel is polymerized with the fixed tissues. Then the plasma membrane of the fixed tissue is removed and the tissue becomes transparent. Finally, immunofluorescent staining is performed ultimately laser skin and confocal microscopy is used to show global structural and cellular changes during biliary degeneration in metamorphic c Lampry.

This method can help answer key questions in the biomedical field, such as the connectomes in the central nervous system, and in our case, the biliary degeneration during serum point metamorphosis. The implications of this technique extend towards therapy or diagnosis of human biliary or atresia because this rare congenital infant disease share similar characteristics with the ary degeneration during serum point metamorphosis. We first had the idea for this method when we found a news article at nature and presented the online video during our lab meeting demonstrating the procedure will be Ann Scott, a graduate student from our laboratory After making phosphate buffered saline and phosphate buffer or pb, according to the text protocol.

In a fume hood, prepare 400 milliliters of hydrogel by combining distilled water, 40%acrylamide 2%bis acrylamide 10 XPBS, 16%paraform, aldehyde, saponin, and ammonium per sulfate. Prepare one liter of clearing solution composed of 4%SDS and 0.2 molar boric acid, and make 500 milliliters of 80%glycerol to prepare tissue in the fume hood. Eloqua 10 milliliters of hydrogel monomer solution into 15 milliliter centrifuge tubes.

Dissect the tissue sample and place the tissue into each tube of hydrogel fix at four degrees Celsius for two days. Following the incubation under a fume hood, polymerize the fixed tissue by adding five microliters of temid. Then mix well and allow the samples to incubate it room temperature for two to three hours.

After the incubation, thoroughly remove the excess gel to clear the tissue samples. Place each in 15 milliliters of clearing solution and incubate at 50 degrees Celsius and 70 RPMs for several days until they become transparent. Change the clearing solution at least once per day and remove any excess gel.

Transfer the samples in 15 milliliters of 0.1 molar PB with 0.1%Triton X 100 at 37 degrees Celsius and 70 RPMs overnight. Repeat the incubation two more times with fresh buffer. Now that the tissue is cleared and free of any excess gel, submerge the samples in primary antibody in PB tritton X 100 and incubate at 37 degrees Celsius and 70 RPMs for two days after the incubation, use 15 milliliters of PB with 0.1%tritton X 100 to wash the tissue samples at 37 degrees Celsius and 70 RPMs overnight.

Repeat the wash three times after removing the final wash buffer, add fluorescent secondary antibody solution in PB with 0.1%Trix 100 and blocking serum and incubate in the dark at 37 degrees Celsius and 70 RPMs for one day. After washing the samples three times in 10 milliliters of PB tritton X 100 at 37 degrees Celsius and 70 RPMs overnight, perform three overnight washes in PBS. Finally incubate the clarified tissue in 80%glycerol covered with foil overnight.

Confocal microscopy imaging is then performed in dark or dim light, though the lights are on in this video for visualization purposes. Several important developmental events occur in the HEPA biliary system during c lampre metamorphosis, for example, as shown here, the bile duct and the gallbladder undergo apoptosis and degenerate. After using the modified clarification method and staining with liver cell marker cytokeratin 19 anti anti apoptotic marker, BCL two laser scanning confocal microscopy was used to perform optical sectioning through the intact liver.

The staining revealed that the degenerative process occurs in metamorphic stages two through four. The top panels are from the anterior and the lower panels are from the posterior end. Shown here is the biliary tree and here are dark dendritic looking apoptotic cells and surrounding canaliculi and duct tools in the liver.

This figure represents three dimensional images reconstructed the confocal Z series of three livers between metamorphic stages two and three labeled with antibodies against cytokeratin 19 and BCL two, the biliary tree and the dark dendritic looking apoptotic cells and surrounding canaliculi and duct tools are marked by white and black arrows respectively. Shown here is a confocal optical section of the bile ducts of a specimen between metamorphic stages two and three with bile droplets in the lumen. After watching this video, you should have a good understanding of how to clarify the entire tissue for further imaging analyses.

Don't forget that working with paraform, aldehyde and poly acrylamide solutions can be extremely hazardous, and precautions such as wearing gloves and using the fume hood should always be taken while performing this procedure.