Layered Agar Mounting: Preparing Live Zebrafish Embryos for Long-Term Imaging with an Inverted Microscope
Published: April 30, 2023
Abstract
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.
Protocol
1. Preparation of embryos After mating, harvest embryos in E3 in a Petri dish and incubate them at 26.5 °C for about 28 h before mounting. NOTE: This slows down the development of the embryos so that the embryos are approximately at 30 somite stage at the beginning of imaging. Anesthetize embryos in 0.016-0.020% Tricaine in E3. To inhibit pigmentation, add PTU to a concentration of 200 µM. Dechorionate the embryos using forceps under a dis…
Representative Results
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) Microscop…
Materials
| 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 |
Bu Makaleden Alıntı Yapın
Layered Agar Mounting: Preparing Live Zebrafish Embryos for Long-Term Imaging with an Inverted Microscope. J. Vis. Exp. (Pending Publication), e20186, doi: (2023).