Dissection of the Adult Zebrafish Kidney

1. Dissection of the adult zebrafish kidney from an unfixed animal sample

1. Select an adult zebrafish between 3-6 months in age for dissection.
2. Euthanize the fish by placing it into a dish with 0.2% Tricaine pH 7.0 for approximately 4-5 minutes, watching carefully to see that the gills stop moving and the heart stops beating.
3. Using a spoon, lift the fish from the Tricaine bath in a small amount of solution, decant the solution and gently place the animal on a paper towel.
4. Use a sharp pair of dissection scissors to remove the head of the animal, making a cut just behind the gill operculum (Figure 1, path A). Discard the head in a biohazard waste container.
5. Use the dissection scissors to open the body of the animal, by making a long ventral incision, starting from the head and terminating at the tail at the base of the caudal fin (Figure 1, path B).
6. Remove the internal organs of the animal using a pair of dissection tweezers/forceps, and place them in biohazard waste. Be careful not to brush the dorsal body wall, as this is the location of the kidney (Figure 2). If you have selected a female fish, you will need to scoop developing eggs out of the body cavity, which can be most easily done using an angled pair of forceps or tweezers.
7. Use dissection needles to pin open the body walls to a dissection tray, and angle the pins so that the kidney can be visualized. The kidney will possess a characteristic shape (head, trunk/saddle, tail) and color, being interspersed with black hued pigmentation (Figure 2). You may also see the aorta filled with peripheral blood, which runs down the midline of the organ structure.
8. Use fine forceps to detach the kidney from the dorsal body wall, using one pair of forceps to lift the kidney as you separate the organ from the connective tissues that underlie it.
9. Gently place the kidney into the desired vehicle for further live cell studies, such as a microcentrifuge tube containing 1x PBS for FACS preparation.

2. Preparation and dissection of the adult zebrafish kidney from a fixed animal sample

1. Prepare a solution of 4% paraformaldehyde/1X PBST. Boil the solution to dissolve the PFA into solution, then cool the mixture to 4°C and add 0.1% dimethylsulfoxide. We have had the most success with tissue preservation from using freshly made PFA solutions as opposed to frozen aliquots. Make enough solution to submerge the adult zebrafish during dissection.
2. Fill a dissection tray with enough fixation solution to submerge the adult zebrafish during the dissection. We perform the subsequent steps with this tray positioned under the microscope, which can be placed in a chemical hood to minimize exposure to fixation odors.
3. Select an adult zebrafish between 3-6 months in age for dissection.
4. Euthanize the fish by placing it into a dish with 0.2% Tricaine pH 7.0 for approximately 5 minutes, watching carefully to see that the gills and heart stop beating.
5. Using a spoon, lift the fish from the Tricaine bath in a small amount of solution, decant the solution and gently place the animal on a paper towel.
6. Use a sharp pair of dissection scissors to remove the head of the animal, making a cut just behind the gill operculum (Figure 1, path A). Discard the head in a biohazard waste container, and immediately place the animal into the PFA-containing dissection tray.
7. Use the dissection scissors to open the body of the animal, by making a long ventral incision, starting from the head and terminating at the tail at the base of the caudal fin (Figure 1, path B), and keep the body partially submerged during the procedure.
8. Remove the internal organs of the animal using a pair of dissection tweezers/forceps, and place them in biohazard waste. Be careful not to brush the dorsal body wall, as this is the location of the kidney (Figure 2).
9. Use dissection needles to pin open the body walls, and angle the pins so that the kidney can be visualized. The kidney will possess a characteristic shape (head, saddle, tail) and color, being interspersed with black hued pigmentation (Figure 2). You may also see the aorta filled with peripheral blood, which runs down the midline of the organ structure.
10. Fix the sample overnight, placing the tray at 4°C when you are done with arranging the desired samples.
11. The next day, remove the PFA solution from the tray and replace with 1x PBST.
12. Use fine forceps to carefully detach the kidney from the dorsal body wall, using one pair of forceps to lift the kidney as you separate the organ from the connective tissues that underlie it. The kidney tissue will be soft and pliable from the DMSO in the fixative solution, making it possible to dissect the entire kidney organ in one piece.
13. Use a wide-bore transfer pipet to place the kidney into a microcentrifuge tube.
14. The kidney can now be processed further to conduct histological or whole mount in situ gene expression studies based on the desired study.

3. Representative results:

The steps that are diagrammed in Figure 1 indicate the dissection procedure. The kidney can be identified based on its characteristic shape and coloration, and anatomical location on the dorsal wall of the animal’s body cavity, as shown in Figure 2. After dissection of a fixed kidney sample, whole mount in situ hybridization can be used to localize the expression of a gene of interest, as shown in Figure 3.

Figure 1: Procedure that enables direct access for dissecting the zebrafish adult kidney. (A) Euthanized zebrafish is dissected in a stepwise fashion (indicated by numbers and arrows) to give the researcher the easiest access to the complete kidney organ. Images of the abdominal cavity before (B) and after (C) removal of the abdominal organs.

Figure 2: Visualization of the zebrafish kidney in an unfixed sample. Following removal of organs in the body cavity, the kidney appears as a single, flattened organ that is adherent to the dorsal body wall via connective tissues (A), and has been schematized (B) to show its anatomical shape.

Figure 3: Whole mount in situ hybridizations performed on the whole adult kidney organ. (A) cadherin17 transcripts are detected in the tubule of adult kidney nephrons, enlarged in (A’). (B) mafba transcripts are localized proximal nephron cell types in the adult kidney (compare to entire tubule in panel A). (B’) An enlarged view of mafba expression reveals that transcripts are specifically localized to the podocyte (P) cells of the blood filter (glomerulus) and proximal convoluted tubule (PCT).