Citation:

Zucker, R. AND J. Rogers. Confocal Laser Scanning Microscopy of Morphology and Apoptosis in Organogenesis-Stage Mouse Embryos. Chapter n/a, Hansen J., Winn L. (ed.), Developmental Toxicology: Methods and Protocols. Springer Nature, New York, NY, , 297-311, (2019). https://doi.org/10.1007/978-1-4939-9182-2_20

Impact/Purpose:

In our efforts to study morphology and apoptosis in organogenesis-stage rodent embryos, we have developed staining, fixation and clearing methods to allow optical sectioning through embryos with thickness approaching one millimeter (z-axis) using confocal laser scanning microscopy. We have combined fixation and clearing methods with fluorochrome staining for several purposes. In this chapter we will present two methods; first, clearing with methyl salicylate (oil of wintergreen) and staining with Nile blue sulfate (NBS, not used as a vital dye for this protocol) for general morphological assessment, and second, staining live embryos with the vital stain LysoTracker Red (LT), followed by fixation and clearing with benzyl alcohol/benzyl benzoate (BABB) to visualize areas of apoptosis. With both protocols, an entire organogenesis-stage rodent embryo can be optically sectioned and reconstructed in three dimensions to reveal areas of dye staining. Emerging methods applicable to such studies will be briefly discussed at the end of this chapter.

Description:

Background: After fluorochromes are incorporated into cells, tissues, and organisms, confocal microscopy can be used to observe three-dimensional structures. LysoTracker Red (LT) is a paraformaldehyde-fixable probe that concentrates into acidic compartments of cells and indicates regions of high lysosomal activity and phagocytosis, both of which correlate to apoptotic activity. Thus, LT is a good indicator of apoptosis visualized by confocal microscopy. Results of LT staining of apoptotic cell death correlate well with other whole mount apoptosis vital dyes such as Nile blue sulfate and neutral red, with the added benefit of being fixable in situ. Nile blue sulfate can also be used as a non-vital, nonspecific dye to visualize general morphology. Stains such as Acridine orange can be used for surface staining of fixed embryos to yield confocal images that are similar to scanning electron micrographs. Methods: Mouse embryos were stained with LT, fixed with paraformaldehyde/glutaraldehyde, dehydrated with methanol (MEOH), and cleared with benzyl alcohol/benzyl benzoate (BABB). Following this treatment, the tissues were nearly transparent. Embryos are mounted on depression slides and optical serial sections are imaged by confocal microscopy, followed by 3-D reconstruction. Alternatively, fixed embryos can be dehydrated, stained with Nile blue sulfate and cleared with methyl salicylate for general morphology, or stained with Acridine orange for opaque surface staining. Results: Embryos or tissues as thick as 500 microns (µm) can be visualized after clearing with BABB. Lysotracker staining reveals apoptotic regions in organogenesis-stage mouse embryos. Morphological observation of tissue was facilitated by combining autofluorescence with Nile blue sulfate staining of fixed embryos or opaque surface staining with Acridine orange. Conclusions: The use of BABB for clearing LT vital-stained and fixed embryos matches the refractive index of the tissue to the suspending medium, allowing increased penetration of laser light in a confocal microscope. Nile blue sulfate used as a non-vital dye provides a nonspecific staining of fixed embryos that can then be cleared with methyl salicylate for confocal observation. Sample preparation and staining procedures described here, with optimization of confocal laser scanning microscopy, allows for the detection and visualization of morphological structure and apoptosis in embryos up to 500 µm thick, and stained specimens can be fixed and mounted on depression slides.

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