kidney flat mount preparation: a procedure to dissect and prepare a flat mount for microscopic analysis of adult zebrafish kidney

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

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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1. Dissection and Flat Mount Preparation of the Adult Zebrafish Kidney from an Unfixed Animal Sample to Visualize Fluorescent Dextran Labeling of the Proximal Tubule
<ol>
	<li>Euthanize the dextran-injected adult zebrafish by placing it into a dish with 0.2% Tricaine pH 7.0 for 4&#8211;5 min. 
	NOTE: Ensure that the fish has been euthanized before proceeding, by monitoring carefully whether the gills have ceased movement and the heart has stopped beating.</li>
	<li>Using a plastic spoon, lift the fish from the Tricaine solution, decant the solution and place the animal on a tissue or paper towel.</li>
	<li>Use a sharp pair of dissection scissors to make a cut behind the gill operculum and remove the head.</li>
	<li>Immediately open the body of the fish with the dissection scissors by making a long ventral incision from the head to the base of the caudal fin.</li>
	<li>Remove the internal organs of the fish using a pair of fine forceps.</li>
	<li>Use super-fine dissection needles to pin open the body walls to allow visualization of the kidney organ, which is adherent to the dorsal wall of the animal.</li>
	<li>Use fine forceps to detach the kidney from the dorsal wall.</li>
	<li>Gently place the kidney into a 5 mL glass vial containing 1X PBS and wash 3 times with 3&#8211;5 mL of fresh 1X PBS for 5 min each. 
	NOTE: The use of a glass vial for handling of adult kidney samples facilitates visualization of the tissue and allows for washes with large volumes (relative to the tissue mass) of approximately 3&#8211;5 mL. Alternatively, a 12-well cell culture dish can be used. While a plastic microcentrifuge tube could be used, the standard sized tubes (1.5 mL) could restrict the washing.</li>
	<li>Remove the kidney with a transfer pipette and place onto a clean glass slide with 1&#8211;2 drops of 1X PBS.</li>
	<li>Use fine forceps, flattened the kidney on the slide, making sure no tissue has curled or otherwise overturned on itself. 
	NOTE: Alternatively, a tungsten wire tool can be used to make small incisions in the connective tissue to facilitate flattened positioning of the kidney.</li>
	<li>Place small pieces of modeling clay on each corner of an 18 x 18 mm glass coverslip and slowly set the coverslip onto the kidney. 
	NOTE: Slightly angle the coverslip during placement to minimize trapping air bubbles in the solution. The modeling clay divots are approximately 2 mm in diameter (Figure 1A). Alternatively, divots of vacuum grease can be substituted in lieu of modeling clay. Add an additional drop of 1X PBS to fill the space between the coverslip and glass slide if necessary.</li>
	<li>Observe and/or image the kidney by placing the glass slide into the field of view on a stereomicroscope or compound microscope with the appropriate filter.</li>
</ol>

<table><tbody><tr><td>1X PBS</td><td></td><td></td><td>Made by diluting 10 X PBS in distilled water.</td></tr><tr><td>Fine Forceps&amp;nbsp;</td><td>Roboz&amp;nbsp;</td><td>RS-1050</td><td>Dumont Tweezers Pattern #55</td></tr><tr><td>Glass slide&amp;nbsp;&amp;nbsp;</td><td>Thermo-Fisher</td><td>4445</td><td>White Frost</td></tr><tr><td>Glass coverslip&amp;nbsp;</td><td>Thermo-Fisher&amp;nbsp;&amp;nbsp;</td><td>12-540A</td><td>18 x 18 mm</td></tr><tr><td>Modeling clay&amp;nbsp;&amp;nbsp;</td><td>Hasbro</td><td>Playdoh</td><td>Other modeling clays can be substituted and work similarly.&amp;nbsp;</td></tr><tr><td>Slide holder&amp;nbsp;&amp;nbsp;</td><td>Thermo-Fisher</td><td>12-587&amp;nbsp;</td><td>Optional: cardboard tray to store slides flat.</td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ645; SMZ1000; 83455 P-Blue GFP/DAPI; 83457 P-Endow GFP/ FITC; 83457 P-TRITC</td><td>Filter set used was as follows: Hoechst/DAPI filter was used for DAPI, propidium iodide, dextran-cascade blue and alkaline phosphatase detection. GFP/FITC filter was used for dextran-FITC, dextran lucifer yellow, and antiGFP detection. The TRITC filter was used for dextran-fluoro-ruby, PI, and DBA detection.</td></tr><tr><td>Compound microscope&amp;nbsp;</td><td>Nikon</td><td>96310 C-FL UV-2E/C DAPI; 96311 C-FL B-2E/C FITC; 96313 C-FL Y-2E/C Texas Red</td><td>Filter set used was as follows: Hoechst/DAPI filter was used for DAPI, propidium iodide, dextran-cascade blue and alkaline phosphatase detection. GFP/FITC filter was used for dextran-FITC, dextran lucifer yellow, and antiGFP detection. The Texas Red filter was used for dextran-fluoro-ruby, PI, and DBA detection.</td></tr></tbody></table>

Source: McCampbell, K. K. et al. <a href="https://www.jove.com/video/51644" target="_blank">Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney.</a>&#160;J. Vis. Exp. (2014)
This video demonstrates the dissection and preparation of a flat mount of the adult zebrafish kidney. The prepared flat mount can be visualized under the microscope for specimen analysis.

<img alt="Figure 1" src="/files/ftp_upload/20573/20573_Figure1.jpg" /> 
Figure 1.&#160;Adult zebrafish kidney flat mount preparation and application to visualize conjugated dextran uptake in the PCT segment of kidney nephrons. (A)&#160;Brightfield image of a kidney specimen flat mount preparation, in which the organ has been positioned flat on a glass slide, with a coverslip placed on top of the tissue that is resting on four divots of modeling clay (hot pink color)....

Source: McCampbell, K. K. et al. Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney.&#160;J. Vis. Exp. (2014)
This video ...

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.&#160;
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.

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

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