Preparation of Precisely Oriented Cryosections of Undistorted Drosophila Wing Imaginal Discs for High Resolution Confocal Imaging   

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Jihyun Kim
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A brief version of this protocol appeared in:
Feb 2018


The combination of immunofluorescence and laser scanning confocal microscopy (LSM) is essential to high-resolution detection of molecular distribution in biological specimens. A frequent limitation is the need to image deep inside a tissue or in a specific plane, which may be inaccessible due to tissue size or shape. Recreating high-resolution 3D images is not possible because the point-spread function of light reduces the resolution in the Z-axis about 3-fold, compared to XY, and light scattering obscures signal deep in the tissue. However, the XY plane of interest can be chosen if embedded samples are precisely oriented and sectioned prior to imaging (Figure 1). Here we describe the preparation of frozen tissue sections of the Drosophila wing imaginal disc, which allows us to obtain high-resolution images throughout the depth of this folded epithelium.

Figure 1. The epithelial structure and undistorted folding pattern are revealed in its entire depth in this frozen section of developing Drosophila wing. A-D. Transverse dorsoventral sections through the wing pouch. A. Cryosection reveals nuclei (A, green) and subcellular distribution of α-catenin (A’, A”, magenta) with signal throughout the depth of the epithelium. The basal surface is clearly detectable (arrows). A” is digitally enhanced image of A’. B. A Z-stack of images collected in a top-down view displayed as XZ orthogonal view reveals nuclei (B) but little discernable detail for α-catenin (B’, B”) and even the digitally enhanced image (B”) fails to reveal the basal epithelial surface (arrow). C. Transverse dorsoventral section displaying the Distal-less (Dll, green) gradient in the wing pouch and subcellular localization of DE-Cadherin (magenta) throughout the epithelium. D. View of the wing pouch. Dorsal is to the left; apical is up. Scale bars are 1 µm in A, B, 11 µm in C, 5 µm in D.

Keywords: Cryosection, Frozen sections, Confocal microscopy, Wing imaginal disc, Drosophila


Third instar imaginal discs are flat pocket-like involutions of the epidermis (Cohen, 1993; McClure and Schubiger, 2005). One layer of this pocket, the ‘disc proper’, is a pseudostratified columnar epithelium that is heavily folded at the onset of metamorphosis. It is continuous with the ‘overlaying’ squamous epithelium, the peripodial membrane. The focus of our work is to understand how the Wnt morphogen patterns the dome-shaped wing pouch region of the wing disc. As imaginal discs are flat overall, conventional imaging has them mounted for top-down or upside-down observation, whereby cover-slips compress and distort the folded structure. The use of spacers prevents distortions, but imaging of the entire wing pouch using Z-stacks has proved unsatisfactory or impossible; as outlined above, the reduced resolution in the Z-axis typically prevents high-resolution reconstruction of the epithelium in the apical/basal direction. Therefore, only the apical half of the epithelium of the wing pouch is detected at high resolution. This problem is exacerbated if weak signals are to be detected. Thus, uniform imaging requires a ‘side-view’ that can be obtained in sections. We modified a cryosection protocol (Culbertson et al., 2011; Sui et al., 2012) to obtain transverse sections of wing discs at defined angles. This methodology was critical to our analysis of signaling gradients in the wing pouch.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Petshow, S. . and Wehrli, M. (2018). Preparation of Precisely Oriented Cryosections of Undistorted Drosophila Wing Imaginal Discs for High Resolution Confocal Imaging. Bio-protocol 8(3): e2725. DOI: 10.21769/BioProtoc.2725.

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