The overall goal of this procedure is to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression, studies of kidney and or blood cell populations. This is accomplished by first selecting a three to six month old adult zebra fish for dissection. The animal is then sacrificed in 0.2%trica with euthanasia being confirmed when the gill stop moving and the heart stops beating.
The third step of the procedure is to use dissection scissors to open the abdominal cavity and to remove the organs located within it. The final step of the procedure is then to use fine dissection forceps to remove the kidney from the underlying connective tissues. Ultimately, gene expression profiles of hematopoetic or kidney cell subsets, isolation of specific cell populations through whole mount.
In situ two hybridization, immunohistochemical analyses and or fluorescence activated cell sorting can be performed on the isolated organ tissue and or cells. Visual demonstration of this method is critical as the dissection steps are difficult to learn. Because a zebrafish kidney is a delicate organ is anchored to the body wall via dense connective tissue.
The implications of this technique can be used in the therapy or diagnosis of kidney and blood diseases in humans, as biological processes are highly conserved between zebrafish and higher mammals. Numerous molecular genetic tools have been developed in recent years in the zebrafish and making it a useful animal model. Poor enough, 0.2%trica to cover an adult zebra fish between three to six months old into a dish.
Then select a zebra fish for dissection and place it in the dish for approximately five minutes, watching carefully to see that the gill stop moving and the heart stops beating. Next, use a spoon to lift the fish outta the trica bath in a small amount of solution. Decant the solution and gently place the animal on a paper towel using a sharp pair of dissection scissors.
Make a cut just behind the gill, a perm to remove the head of the animal, discard the head in a biohazard waste container. Then starting from the head, use the dissection scissors to make a long ventral incision that terminates at the tail to open the body of the animal. Now remove the internal organs of the animal using a pair of dissection tweezers and place them in the biohazard waste.
Be careful not to brush the dorsal body wall as this is the location of the kidney. If you have selected a female fish, scoop the developing X outta the body cavity with an angled pair of forceps or tweezers. Use dissection needles to pin the body walls to a dissection tray.
Angle the pins so that the kidney can be visualized. The kidney possesses a characteristic shape with a head, a trunk or saddle, a tail, and a characteristic color being interspersed with black hued pigmentation. Use two pairs of fine forceps to detach the kidney from the dorsal body wall.
Use one pair to lift the kidney and one pair to hold the underlying connective tissue. As you remove the organ from the zebrafish body cavity, gently place the kidney into the desired vehicle For further live cell studies, such as a micro centri use tube containing one times PBS. For fax preparation, fill a dissection tray with enough freshly prepared fixation solution to submerge a three to six month old zebra fish during a dissection.
Then pour enough 0.02%trica pH seven into a dish. To cover the zebra fish, select a zebra fish for dissection and place it into the dish of trica for euthanasia. Move and discard the head of the animal as shown previously, but this time immediately place the animal into the PFA containing dissection tray.
After the removal, keeping the body partially submerged in the fixative during the procedure, use dissection scissors to open the body of the animal with a ventral incision as before. Again, being careful not to brush the dorsal body wall, remove the internal organs of the animal using a pair of dissection tweezers. Place the organs in a biohazard waste container.
As before, use dissection needles to pin open the body walls, angling the pins so that the kidney can be visualized. Then fix the sample overnight. Placing the tray at four degrees Celsius the next day, remove the fixative solution from the tray and replace with one times PBST.
Then, as before, carefully detach the kidney from the dorsal body wall using one pair of forceps to lift the entire kidney organ in one piece as you separate it from the connective tissues that underlie it. Next, use a wide bore transfer pipette to place the kidney into a micro centrifuge tube for further processing. As shown here, following the of organs in the body cavity, the kidney appears as a single flattened organ that is a adherent to the dorsal body wall via connective tissues.
In this figure, the kidney has been schematic size to show its characteristic anatomical shape. Here kadin 17 transcripts detected by whole mountain C two hybridization of the kidney can be seen in the tubule of adult kidney nephrons with an enlarged view of the tubule staining shown in the right panel. In this figure, it can be seen in the left panel that M-A-F-B-A transcripts a localized to proximal nephron cell types in the adult kidney.
The enlarged view of M-A-F-B-A expression as visualized on the right reveals that the transcripts are specifically localized to the podocyte cells of the gliomas as indicated by the p and arrow and to the proximal convoluted tubule indicated by the PCT and bracket. While attempting this procedure, it's important to remember to remove the internal organs of the adult zebra fish with care so not to damage the kidney prior to its surgical isolation Following this procedure. Other methods such as histology or fluorescence activated cell sorting can be used to assess the ultra structural characteristics of blood and kidney cell populations or to isolate specific populations for cell transplantation or primary cell culture.
