Kidney Flat Mount Preparation: A Procedure to Dissect and Prepare a Flat Mount for Microscopic Analysis of Adult Zebrafish Kidney

JoVE Encyclopedia of Experiments

Kankeronderzoek

0 weergaven • 2:57 min • April 30th, 2023

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The adult zebrafish contains a single, flattened kidney attached to the dorsal body wall through connective tissues. It consists of three main regions: head, trunk, and tail. The functional unit of the kidney, nephrons, are highly branched and drain into two major collecting ducts.

To isolate the kidney, first, prep a euthanized adult zebrafish with its ventral side facing up. Make an incision behind the operculum - a bony flap covering the gills - to remove the head. Subsequently, make another incision from the head to the base of the caudal or tail fin to expose and remove the internal organs.

Now, pin open the body walls to visualize the kidney. Dissect the underlying connective tissue to detach the kidney from the dorsal wall. Transfer the organ into a vial containing a suitable buffer, washing it thoroughly to remove debris. The buffer helps provide physiological conditions. Post wash, place the kidney, hydrated with buffer, on a glass slide.

Ensure that the kidney is completely flattened, making cuts in the connective tissue if necessary. This step aids in easy visualization during imaging. Finally, secure the organ preparation using a coverslip affixed with modeling clay pieces at its corners. The clay helps prevent tissue deformation. The flat-mounted tissue is now ready for microscopic analysis.

To begin, decant away any extra solution from an anesthetized fish and place it on a tissue or paper towel. After removing the head and internal organs, use superfine dissection needles to pin open the body walls. Locate the kidney organ, which is adherent to the dorsal wall of the animal.

Use fine forceps to detach the kidney from the dorsal wall. Gently place the kidney into a 5-milliliter glass vial containing 1x PBS and wash three times for 5 minutes each. Remove the kidney with a transfer pipette and place it onto a clean glass slide with one to two drops of 1x PBS.

Use fine forceps to flatten the kidney on the slide, making sure no tissue has curled or otherwise overturned on itself. A tungsten wire tool can be used to make small incisions in the connective tissue to facilitate the flat positioning of the kidney.

Next, place small pieces of modeling clay on each corner of an 18 by 18 millimeter glass coverslip. Slowly set the coverslip onto the kidney, keeping a slight angle to minimize trapping air bubbles. Add an additional drop of 1x PBS to fill the space between the coverslip and slide if necessary.

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Laatst bijgewerkt: 15 augustus 2026