This article describes the process of preparing the adult zebrafish kidney for staining by depigmenting it to enhance visualization. The methodology includes dissection, treatment with sucrose solutions, and bleaching to remove melanocyte pigmentation.
The adult zebrafish kidney is a translucent structure located adjacent to the dorsal body wall of the fish. The kidney exhibits the presence of superficial black spots corresponding to the scattered melanocytes - specialized cells that protect the kidney stem cells from harmful UV radiations.
To depigment and prepare the kidney for staining, begin by taking a euthanized adult zebrafish. Submerge the whole zebrafish in a suitable fixative solution and incubate for the desired duration. Incubation allows the fixative solution to penetrate the fish's body and lock the tissue components in place.
Next, place the zebrafish ventrally and remove its head. Make a long ventral incision starting beneath the head region and extending till the caudal fin. Remove the organs to expose the underlying kidney. Using forceps, detach the kidney from the dorsal wall and place it in a glass vial.
Treat the dissected kidney with an increasing concentration of sucrose solution. Sucrose solution creates an isotonic environment that prevents kidney shrinkage. Subsequently, incubate the kidney in a bleaching solution to remove melanocyte pigmentation. This helps in better visualization of the kidney during further staining procedures.
Finally, replace the bleaching solution with a blocking solution to block unwanted sites and prevent any nonspecific staining. The depigmented adult zebrafish kidney is now ready for subsequent staining steps.
When ready, use fine forceps to carefully detach the kidney from the dorsal body wall and place the organ into a glass vial. Wash the dissected kidney three times with 3 to 5 milliliters of 1x PBS with 0.05% Tween for 5 minutes each.
Next, remove the PBS solution and rinse the kidney with 3 milliliters of a 5% sucrose solution for 30 minutes. After this time, replace the solution with 3 milliliters of 30% sucrose and store overnight at 4 degrees Celsius.
The following day, remove the solution and wash the kidney two times before adding 3 to 5 milliliters of bleaching solution to remove the melanocyte pigmentation present on the kidney organ. Place the glass vial on a rotator and watch carefully as the pigmentation disappears. Depigmentation typically takes approximately 20 minutes but occasionally can take as long as 60 minutes.
If the bleaching solution is left on too long, disintegration can occur, and it is therefore advised to monitor the sample every 10 to 15 minutes to check tissue integrity. When the pigmentation has been removed from the kidney, wash twice and incubate with 4% PFA solution for 1 hour at room temperature.
Next, remove the 4% PFA solution and wash the kidney three times. After the last wash, add 3 to 5 milliliters of blocking solution and incubate the kidney at room temperature for 2 hours. When blocking is complete, proceed directly to the selected staining protocol.
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