Source: Upadhyay S., et.al. A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos. J. Vis. Exp. (2020).
This article describes a method to mount live zebrafish embryos for long term imaging. This method is cost effective and easy to perform using regular glass-bottom microscopy dishes for imaging on Inverted Microscope. The mounting is performed in layers of agarose at different concentrations.
1. Preparation of embryos
2. Mounting in agarose
NOTE: The developed mounting method requires two different concentrations of low-melt agarose in E3 with 0.02% Tricaine and PTU as needed. The first agarose solution contains an optimal concentration of agarose at which the distortion and motility are at a minimum. The optimization is described in step 5 below.
3. Optimization of agarose solution for layer 1
Figure 1 : Description of mounting method. (A) Add the zebrafish embryo to the small well created by the glass bottom in the 35 mm dish. (B) Add agarose layer 1 to the small well to cover the embryo. (C) Carefully place a cover glass over the small well. (D) Add agarose layer 2 on the whole bottom of the 35 mm dish. (E) Add E3 to the dish. (F) Schematic drawing of a cross section of the mounting set up. (G) Microscope image (5x objective) of the zebrafish embryo in the final montage.
Low melting agarose | Sigma-Aldrich, MO | A9414 | Store dissolved solution at 4 °C |
35 mm glass bottom dishes with No. 0 coverslip and 10 mm diameter of glass bottom | MatTek Corporation, MA | P35GCOL-0-10-C | |
Tricaine (MS-222) | Sigma-Aldrich, MO | E10521 | Store dissolved solution at 4 °C |
N-phenylthiourea (PTU) | Sigma-Aldrich, MO | P7629 | Store dissolved solution at -20 °C |
DMC4500 digital microscope camera | Leica | NA | |
Micro cover glass 22×22 mm | VWR | 48366 067 |