Imaging the Dorsal-Ventral Axis of Live and Fixed Drosophila melanogaster Embryos

Abstract

Optimal imaging conditions are of critical importance in developmental biology, as much of the data in the discipline is acquired through microscopy. However, imaging deep sections of tissue, especially live tissue, can be a technical challenge due to light scattering and difficulties in mounting the sample. In particular, capturing high-quality images of dorsal-ventral cross sections requires "end-on" mounting to orient the anterior-posterior axis vertically. Here we present methods to mount and image dorsal-ventral cross sections of both live and fixed Drosophila melanogaster embryos. Our methods have the advantages of being rapid, allowing deep optical sections, and not requiring expensive, specialized equipment.

A facilitated diffusion mechanism establishes the Drosophila Dorsal gradient

“…Embryos were cross-sectioned and mounted in 70% glycerol as described previously (Carrell and Reeves, 2015). Briefly, a razor blade was used to remove the anterior and posterior thirds of the embryo, leaving a cross section ∼200 µm long by 200 µm in diameter.…”

A Facilitated Diffusion Mechanism Establishes the Drosophila Dorsal Gradient

“…Embryos were cross sectioned and mounted in 70% glycerol as described previously (Carrell and Reeves, 2015). Briefly, a razor blade was used to remove the anterior and posterior thirds of the embryo, leaving a cross section roughly 200 µm long by 200 µm in diameter.…”

Dynamics of BMP signaling in the earlyDrosophilaembryo

“…Nc 14 embryos expressing Med-GFP and immunostained against pMad and GFP were cross sectioned using a razor by removing the anterior and posterior thirds of the embryo, as described previously (Carrell and Reeves, 2015). The cross sections were mounted in 70% glycerol and imaged using a Zeiss LSM 710 microscope at 20x magnification.…”

“…The coverslip was then adhered with double-sided tape to a 21.5 mm-tall steel mounting block in the shape of a half cylinder with a 30 mm diameter (Carrell and Reeves, 2015). The short edge of the coverslip to which embryos were not adhered was aligned with the top of the mounting block, such that 0.5 mm of the coverslip (to which embryos were adhered) was extending past the bottom of the mounting block (Carrell and Reeves, 2015). The mounting block was then placed into a glass-bottom 35 mm petri dish with No.…”

“…To measure the dynamics of nuclear Med-GFP, we imaged live embryos (n = 2) end-on with a confocal microscope roughly 100 μm from the posterior pole (Figs. 3A-C) (Carrell and Reeves, 2015). Expression of H2A-RFP allowed us to segment the nuclei to distinguish nuclear from cytoplasmic pixels.…”