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

Abstract

Source: McCampbell, K. K. et al. Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney. 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.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 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 Euthanize the dextran-injected adult zebrafish by placing it into a dish with 0.2% Tricaine pH 7.0 for 4–5 min. NOTE: Ensure tha…

Representative Results

Figure 1. Adult zebrafish kidney flat mount preparation and application to visualize conjugated dextran uptake in the PCT segment of kidney nephrons. (A) 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)….

Materials

1X PBS			Made by diluting 10 X PBS in distilled water.
Fine Forceps	Roboz	RS-1050	Dumont Tweezers Pattern #55
Glass slide	Thermo-Fisher	4445	White Frost
Glass coverslip	Thermo-Fisher	12-540A	18 x 18 mm
Modeling clay	Hasbro	Playdoh	Other modeling clays can be substituted and work similarly.
Slide holder	Thermo-Fisher	12-587	Optional: cardboard tray to store slides flat.
Stereomicroscope	Nikon	SMZ645; SMZ1000; 83455 P-Blue GFP/DAPI; 83457 P-Endow GFP/ FITC; 83457 P-TRITC	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.
Compound microscope	Nikon	96310 C-FL UV-2E/C DAPI; 96311 C-FL B-2E/C FITC; 96313 C-FL Y-2E/C Texas Red	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.