Light Sheet Microscopy Sample Preparation: Mounting Live Zebrafish Embryos for Long-Term Imaging
Published: April 30, 2023
Abstract
Source: Icha J., et al. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development. J. Vis. Exp. (2016).
This article describes sample preparation for light-sheet fluorescence microscopy. It also demonstrates a method to mount live zebrafish embryos for long term imaging, which allows recording of long time-lapse movies of live embryos in near-physiological conditions.
Protocol
1. Sample Preparation Preparing the Agarose Mix 15 min before making of the agarose mix, melt one 1 ml aliquot of 1% low melting point agarose (dissolved in E3 medium) in a heating block set to 70 °C. Once the agarose is completely molten, transfer 600 µl into a fresh 1.5 ml tube, add 250 µl of E3 medium, 50 µl of 0.4% MS-222 and 25 µl of vortexed bead stock solution. NOTE: This makes 925 µl of the mix, while additional 75 …
Materials
| Lightsheet Z.1 microscope | Carl Zeiss Microscopy | ||
| Low melting point agarose | Roth | 6351.1 | |
| Low melting point agarose | Sigma | A4018 or A9414 | |
| Ethyl 3-aminobenzoate methanesulfonate (MESAB/ MS-222/Tricaine) | Sigma | E10521 | |
| 20 μl (1 mm inner diameter, marked black) capilllaries | Brand | 701904 | sold as spare part for transferpettor |
| Teflon tip plungers for 20 μl capillarie | Brand | 701932 | sold as spare part for transferpettor |
