Skip to main content
SpringerLink
Log in

Confocal Laser Scanning Microscopy of Morphology and Apoptosis in Organogenesis-Stage Mouse Embryos

  • Protocol
  • First Online:
Developmental Toxicology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1965))

Abstract

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 serial sections are imaged by confocal microscopy, followed by 3-D reconstruction.

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 staining.

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, allow 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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Zucker RM, Hunter S, Rogers JM (1998) Confocal laser scanning microscopy of apoptosis in organogenesis-stage mouse embryos. Cytometry 33:348–354

    Article  CAS  Google Scholar 

  2. Sulik KK, Dehart DB, Johnson C, Ellis SL, Chen SY, Dunty WC, Zucker RM (2001) Programmed cell death in extraocular muscle tendon/sclera precursors. Mol Vis 7:184–191

    CAS  PubMed  Google Scholar 

  3. Dunty WC, Zucker RM, Sulik KK (2002) Hindbrain and cranial nerve dysmorphogenesis result from acute maternal ethanol administration. Dev Neurosci 24:328–342

    Article  CAS  Google Scholar 

  4. Dunty WC, Chen SY, Zucker RM, Dehart DB, Sulik KK (2001) Selective vulnerability of embryonic cell populations to ethanol-induced apoptosis: implications for alcohol related birth defects and neurodevelopmental disorder. Alcohol Clin Exp Res 25:1523–1535

    Article  CAS  Google Scholar 

  5. Watanabe M, Hitomi M, van der Wee K, Rothenberg F, Fisher SA, Zucker RM, Svoboda KH, Goldsmith EC, Heiskanen KM, Nieminen AL (2002) The pros and cons of apoptosis assays for use in the study of cells, tissues and organs. J Microsc 8:375–391

    CAS  Google Scholar 

  6. Dunty WC, Chen SY, Zucker RM, Dehart DB, Sulik KK (2001) Selective vulnerability of and neurodevelopmental disorder alcoholism. Clin Exp Res 25:1523–1535

    Article  CAS  Google Scholar 

  7. Rogers JM, Brannen KC, Barbee BD, Zucker RM, Degitz SJ (2004) Methanol exposure during gastrulation causes holoprosencephaly, facial dysgenesis, and cervical vertebral malformations in C57BL/6J mice. Birth Defects Res Part B Dev Reprod Toxicol 71:80–88

    Article  CAS  Google Scholar 

  8. Price OT, Lau C, Zucker RM (2003) Evaluation of apoptosis in fetal rat limbs using confocal laser scanning microscopy and quantitative fluorescence techniques. Cytometry 53A:9–21

    Article  Google Scholar 

  9. Zucker RM (2006) Technical note: whole insects and mammalian embryo imaging with confocal microscopy: morphology and apoptosis. Cytometry 69A:1143–1152

    Article  Google Scholar 

  10. Zucker RM, Jeffay SC (2006) Confocal laser scanning microscopy of whole mouse ovaries: excellent morphology with apoptosis detection and spectroscopy. Cytometry 69A:930–939

    Article  Google Scholar 

  11. Rogers JM, Francis BM, Sulik KK, Alles AJ, Massaro EJ, Zucker RM, Elstein KH, Rosen MB, Chernoff N (1994) Cell death and cell cycle perturbation in the developmental toxicity of the demethylating agent, 5-aza-2′-deoxycytidine. Teratology 50:332–339

    Article  CAS  Google Scholar 

  12. Chernoff N, Rogers JM, Alles AJ, Zucker RM, Elstein KH, Massaro EJ, Sulik KK (1989) Cell cycle alterations and cell death in cyclophosphamide teratogenesis. Teratog Carcinog Mutagen 9:199–209

    Article  CAS  Google Scholar 

  13. Saunders JW Jr, Gasseling MT (1962) Cellular death in morphogenesis of the avian wing. Dev Biol 5:147–178

    Article  Google Scholar 

  14. Abrams JM, White K, Fessler LI, Steller H (1993) Programmed death during drosophila embryogenesis. Development 117:29–43

    CAS  Google Scholar 

  15. Saunders JW Jr (1966) Death in embryonic systems. Science 154:604–612

    Article  Google Scholar 

  16. Alles AJ, Sulik KK (1993) A review of caudal dysgenesis and its pathogenesis as illustrated in an animal model. Birth Defects Orig Artic Ser 29:83–102

    CAS  PubMed  Google Scholar 

  17. Johnson CS, Zucker RM, Hunter ES 3rd, Sulik KK (2007) Perturbation of retinoic acid (RA)-mediated limb development suggests a role for diminished RA signaling in the teratogenesis of ethanol. Birth Defects Res A Clin Mol Teratol 79:631–641

    Article  CAS  Google Scholar 

  18. Menkes B, Prelipceanu O, Capalnasan I (1979) Vital fluorochroming as a tool for embryonic cell death research. In advances in the study of birth defects vol. 3 Abnormal Embryogenesis TVN Persaud ed University Park Press. Balt For 3:219–241

    Google Scholar 

  19. Philips FS, Sternberg SS, Schwartz HS, Cronin AP, Sodergrenae AE, Vidal PM (1967) Hydroxyurea acute cell death in proliferating tissues in rats. Cancer Res 27:61–74

    CAS  PubMed  Google Scholar 

  20. Rogers JM, Taubeneck MW, Dalston GP, Sulik KK, Zucker RM, Elstein KH, Jankowski MA, Keen CL (1995) Zinc deficiency causes apoptosis but not cell cycle alterations in organogenesis-stage rat embryos: effect of varying duration of deficiency. Teratology 52:149–159

    Article  CAS  Google Scholar 

  21. Takahashi M, Osumi N (2010) The method of rodent whole embryo culture using the rota tor-type bottle culture system. J Vis Exp 42:2170

    Google Scholar 

  22. Paddock SW (1994) To boldly glow … applications of laser scanning confocal microscopy in developmental biology. BioEssays 16:357–365

    Article  CAS  Google Scholar 

  23. White NS, Errington RJ, Fricker MD, Wood JL (1996) Aberration control in quantitative imaging of botanical specimens by multidimensional fluorescence microscopy. J Microsc 181:99–116

    Article  Google Scholar 

  24. Pawley JB (1995) Handbook of biological confocal microscopy. Plenum Press, New York

    Book  Google Scholar 

  25. Gard DL (1993) Confocal immunofluorescence microscopy of microtubules in amphibian oocytes and eggs. Methods Cell Biol 38:231–264

    Google Scholar 

  26. Klymkowsky MW, Hanken J (1991) Whole mount staining of Xenopus and other vertebrates. Methods Cell Biol 36:419–441

    Article  CAS  Google Scholar 

  27. Maziere AM, Hage WJ, Ubbels GA (1996) A method for staining of cell nuclei in Xenopus laevis embryos with cyanine dyes for whole-mount confocal laser scanning microscopy. J Histochem Cytochem 44:399–402 15

    Article  Google Scholar 

  28. Zucker RM, Price OT (2001) Evaluation of confocal system performance. Cytometry 44:273–294

    Article  CAS  Google Scholar 

  29. Zucker RM, Price OT (2001) Statistical evaluation of confocal microscopy images. Cytometry 44:295–308

    Article  CAS  Google Scholar 

  30. Zucker RM, Rigby P, Clements I, Salmon W, Chua M (2007) Reliability of confocal spectral imaging systems: use of multispectral beads. Cytometry 71A:174–189

    Article  Google Scholar 

  31. Zucker RM (2006) Confocal slide based system: performance. Cytometry 69A:659–676

    Article  Google Scholar 

  32. Zucker RM (2006) Confocal microscopy slide based systems: instability. Cytometry 69A:677–690

    Article  Google Scholar 

  33. Richardson DS, Lichtman JW (2015) Clarifying tissue clearing. Cell 162:246–257

    Article  CAS  Google Scholar 

  34. Seo J, Choe M, Kim SY (2016) Clearing and labeling techniques for large-scale biological tissues. Mol Cell 39:439–446

    Article  CAS  Google Scholar 

  35. Susaki EA, Ueda HR (2016) Whole-body and whole-organ clearing and imaging techniques with single-cell resolution: toward organism-level systems biology in mammals. Cell Chem Biol 23:137–157

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Disclaimer: The research described in this article has been reviewed and approved for publication as an EPA document. Approval does not necessarily signify that the contents reflect the views and policies of the agency nor does the mention of trade names or commercial products constitute endorsement or recommendation for use.

Author information

Authors and Affiliations

  1. Toxicity Assessment Division, National Health and Environmental Effects Research Laboratory, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, NC, USA

    Robert M. Zucker & John M. Rogers

Authors
  1. Robert M. Zucker
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John M. Rogers
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robert M. Zucker .

Editor information

Editors and Affiliations

  1. Department of Physiology and Developmental Biology, Brigham Young University, Provo, UT, USA

    Jason M. Hansen

  2. Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada

    Louise M. Winn

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Zucker, R.M., Rogers, J.M. (2019). Confocal Laser Scanning Microscopy of Morphology and Apoptosis in Organogenesis-Stage Mouse Embryos. In: Hansen, J., Winn, L. (eds) Developmental Toxicology. Methods in Molecular Biology, vol 1965. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9182-2_20

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9182-2_20

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9181-5

  • Online ISBN: 978-1-4939-9182-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation