Techniques for Rapidly Sampling Six Crucial Organs in Adult Xenopus

Prior to this work, I developed a protocol for rapid amphibian perfusion. This protocol should be utilized before sampling for research such as proteomics and immunochemistry, where the presence of excessive amounts of blood in tissues is undesirable. Though there are many diagrams and other aids available, there are currently no comprehensive guides on how to dissect tissues out of adult Xenopus rapidly and reliably.

We hope that having standardized sampling techniques will allow labs to easily share tissues. This is especially advantageous in situations where importing and exporting animals is not possible due to biosecurity concerns or shipping constraints. To begin, take the frog that has been immersed in the euthanasia solution for one hour, and pinch its foot to check for the absence of a pain response.

Weigh the Xenopus and take additional measurements such as the length from snout to vent. Place the frog on its back, and secure its limbs close to the body. Using dissection scissors, make an incision through the skin along the midline, and then extend it laterally to create two skin flaps.

Then locate the linea alba and grasp it with forceps. Gently pull it from the coelomic cavity and, using scissors, cut through the underlying musculature. Create two flaps from the cavity wall.

Identify the still-beating heart and use dissecting scissors to trim the cricoid bones for better access. Now, employ tissue forceps to identify the thin pericardium and stretch it. Using the tip of iridectomy scissors, carefully perforate the pericardium.

Then, remove the pericardium away from the heart's three chambers. In this protocol, the sampling of the heart ventricle, left lobe of liver, pancreas, fat body, paired kidneys are shown. To sample the heart ventricle, grasp the ventricle at the apex with forceps, locate its connection to the oracles and arterial trunk, and sever it below these points.

Next, observe the three lobes of the liver. Lift the left lobe gently to expose the hepatic and cystic ducts, and sample one third of this lobe below the ducts. If the frog is female, locate the ovary covered by the germinal epithelium.

Gently reposition the lobes to each side to reveal the area of attachment. Use scissors to excise the ovaries as close to the kidneys as possible without damaging them. Then, examine the medial lobe of the liver and its connections to the stomach and duodenum through the mesentery and the hepatopancreatic duct.

Using iridectomy scissors, cut the mesentery hepatoduodenal ligament and hepatopancreatic duct near the duodenum. Detach the pancreas and duct from the liver's medial lobe without leaving dark liver tissue attached. Next, hold the stomach with the help of toothed forceps.

Then, use tissue forceps to hold the pancreas superior end. Under 5X magnification, carefully separate the pancreas from the stomach. Now, locate and excise the urinary bladder, cutting as close to the cloaca as possible, and then discard the tissue.

After that, identify the large intestine and stretch it, severing it near the cloaca. Next, separate the fat bodies to each side. Grasp the base of the left fat body and cut it from the peritoneum.

In cases of female frogs or males with visible vestigial oviducts, hold an oviduct and separate it from the kidney and cloaca. Cut at the cloaca junction and continue to detach it from the kidney, removing any clear peritoneal attachments. Now, use forceps to hold the kidneys and cut the peritoneum at their lower end.

Lift the kidneys from the coelomic cavity. Using scissors, detach the peritoneum as close to the kidneys as possible without causing harm. Under 5X magnification, remove any excess peritoneum and residual tissues such as testis, fat bodies, or spleen.

After releasing the animal, place it on its stomach and secure its limbs. Choose one hind limb for sampling and secure its foot. Excise an almond-shaped skin flap from over the gastrocnemius or tibio-fibula area.

Afterward, rinse all samples and PBS, check for excess tissues, and trim before use if required. The heart ventricle, the left lobe of the liver, the pancreas, the left fat bodies, paired kidneys, and a flap of skin were cleanly excised within an hour of euthanasia.