The overall goal of this procedure is to generate a three dimensional cast of a vascular or ductal tissue. This is accomplished by first preparing and cannulating the target structure, such as the portal vein, and tying the cannula in place. Next, resin is prepared, injected into the portal vein and allowed to cure.
Then the liver is removed and the tissue is fixed or macerated. Finally, the liver is cleared and the cast structure is visualized. Ultimately, results can be obtained.
That show changes in the architecture of the vascular or ductal structure through visual analysis or quantification by micro ct. Though this method can provide insight into liver architecture. It can also be applied to the vascular or ductal systems of any organ, including the brain, lung, pancreas, and kidneys.
To prepare the cannula with your fingertips warm a one inch long section of PE 10 tubing and gently pull it so that it becomes thin. Cut the stretched tubing at a diagonal. To generate a beveled tip, cut the other edge of the tubing to create a five to six inch segment.
Insert a 32 gauge half inch hypodermic needle into the non beveled edge of the tubing. Prepare a three milliliter syringe by first filling it with PBS and screw on the needle with the tubing. Ensure that there are no air bubbles in the syringe, and that the needle is filled with PBS by flicking the syringe and pushing the plunger.
Weigh out 0.1 grams of catalyst into a small container. Then pull one milliliter of resin into a three milliliter syringe. Finally, cut a five inch piece of silk suture to be used as a ligature.
After sacrificing an adult mouse, lay it on its back and open up the abdominal cavity. Lay the mouse on the platform of a dissecting microscope and position a 15 milliliter conical tube perpendicularly under the mouse to increase visibility and accessibility to the liver. Wet a cotton swab with PBS and use it to flip the liver upwards and away to expose the dorsal view of the liver along with the extra hepatic portal, vein, or common bile duct.
For portal vein casts prevent blood clotting by flushing the vein with room temperature. PBS attach a needle to a three milliliter syringe filled with PBS and as far from the liver as possible. Insert it into the extra hepatic portal vein.
Next, gently push PBS into the portal vein. Then make a nick in the common cardinal vein to drain blood if necessary to enhance blood drainage, use a wet cotton swab to massage the liver. If working with an open-ended system, it may be helpful to tie and tighten a ligature at the other end of the system.
If the common cardinal vein is opened to drain blood from the portal vein to tie a ligature around it to increase pressure within the portal vein and prevent resin leakage into the central hepatic vein. Use curved forceps to pass the surgical suture ligature underneath either structure approximately one quarter inch from the liver. Tie the suture into a loose common knot without tightening it.
Next, use spring scissors to make a small cut in the portal vein or bile duct approximately one quarter inch below the suture knot. This cut should penetrate no more than halfway through the diameter of either structure. Then using number five forceps, hold the portal vein or bile duct at the site of the cut and insert the beveled end of the tubing.
Push the tip of the tubing past the prettied ligature towards the liver. Test for cannulation accuracy by injecting PBS through the cannula and watching to see if it enters the portal vein or bile duct. Tighten the ligature to hold the cannula in place.
Add the premeasured one milliliter of resin to the premeasured 0.1 grams of catalyst and mix thoroughly avoiding air bubbles. Draw the resin catalyst mixture back into the three milliliter syringe. Remove any air bubbles by gently flicking the syringe and expelling bubbles.
Carefully replace the PBS containing syringe with the resin containing syringe, leaving the 32 gauge needle attached to the cannula. Slowly and gently push resin into the portal vein or bile duct until resistance increases and the liver is filled. To encourage an even fill.
Use a wet cotton swab to gently massage the liver, remove the cannula, and quickly retighten the ligature To prevent resin leakage. Allow the mouse to lie flat at room temperature for 20 minutes while the resin cures before removing the liver to clear the liver for inside two visualization, fix it in 4%Paraform aldehyde overnight at four degrees Celsius the next day. Pour off the and add PBS.
Wash the liver in PBS for at least four hours. The liver lobes may be separated at this point if desired. Dehydrate the tissue in 50%methanol and then in 100%methanol for at least four hours each.
Rocking at room temperature. Clear the liver in a one to two solution of benzoyl alcohol and benzoyl benzoate for at least 24 hours longer. Wash times may be needed for imaging in situ.
Submerge the liver in BABB in a glass petri dish as an alternative method to macerate the tissue. After removing the liver from the animal, place it into a 50 milliliter conical tube with water. Wait one hour for complete resin curing.
Pour off the water and move the liver to a glass bottle. Submerge it in 15%potassium hydroxide, and leave it overnight at room temperature without rocking carefully. Pour off the potassium hydroxide and wash the cast in distilled water.
Avoid disrupting the cast when adding or removing fluid from the tube. When the resin cast is clean, gently lay it on a kim wipe to dry, then transfer it to a container to be stored. This figure shows a portal vein cast as visualized in situ after BABB clearing.
Shown here is the cast of the entire left liver lobe and the shape and size of the cast. This is a closeup view of the same cast demonstrating the penetration of the resin into the smallest portal vein branches at the liver periphery. Here, a portal vein cast has undergone potassium hydroxide maceration with the entire left lobe shown here, and a closeup on the small peripheral branches here.
Common problems include incomplete fills, bubbles in the resin and overflow into surrounding systems or tissues. This figure demonstrates how to recognize an incomplete fill bubbles and an overflow of resin from the portal vein to the central vein, Don't forget that working with resin can be extremely hazardous and taking precautions such as always wearing gloves and working in a well-ventilated area should be used when performing this procedure.
