Protocol | Published:

Ex vivo time-lapse confocal imaging of the mouse embryo aorta

Nature Protocols volume 6, pages 17921805 (2011) | Download Citation

Abstract

Time-lapse confocal microscopy of mouse embryo slices was developed to access and image the living aorta. In this paper, we explain how to label all hematopoietic and endothelial cells inside the intact mouse aorta with fluorescent directly labeled antibodies. Then we describe the technique to cut nonfixed labeled embryos into thick slices that are further imaged by time-lapse confocal imaging. This approach allows direct observation of the dynamic cell behavior in the living aorta, which was previously inaccessible because of its location deep inside the opaque mouse embryo. In particular, this approach is sensitive enough to allow the experimenter to witness the transition from endothelial cells into hematopoietic stem/progenitor cells in the aorta, the first site of hematopoietic stem cell generation during development. The protocol can be applied to observe other embryonic sites throughout mouse development. A complete experiment requires 2 d of practical work.

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Acknowledgements

We thank the 'Experimentele Medische Instrumentatie' Department of the Erasmus Medical Center and P. Hartwijk for building the black round plastic disks. We also thank R. Koppenol from Cluster 15 of the Erasmus Medical Center for the pictures. This work was supported by NWO (Vidi Dutch young investigator grant [917-76-345]). We thank N. Galjart for careful reading of the manuscript.

Author information

Affiliations

  1. Erasmus Medical Center, Department of Cell Biology, Rotterdam, The Netherlands.

    • Jean-Charles Boisset
    • , Charlotte Andrieu-Soler
    • , Thomas Clapes
    •  & Catherine Robin
  2. Erasmus Medical Center, Erasmus Stem Cell Institute, Rotterdam, The Netherlands.

    • Jean-Charles Boisset
    • , Thomas Clapes
    •  & Catherine Robin
  3. Institut National pour la Santé et la Recherche Médicale (INSERM) UMRS872, Physiopathology of Ocular Diseases: Therapeutic Innovations, Paris, France.

    • Charlotte Andrieu-Soler
  4. Erasmus Medical Center, Department of Reproduction and Development, Erasmus Optical Imaging Centre, Rotterdam, The Netherlands.

    • Wiggert A van Cappellen

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Contributions

C.R. and C.A.-S. developed the nonfixed embryo slicing. C.R., J.-C.B. and W.A.v.C. developed the time-lapse confocal imaging procedure of nonfixed embryo slices/embryo caudal parts. C.R. and J.-C.B. developed and performed the described protocol. All authors wrote the paper. T.C. filmed and edited the three movies showing the experimental procedure.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Catherine Robin.

Supplementary information

Image files

  1. 1.

    Supplementary Fig. 1

    Multicolor staining of embryo slices with directly labeled antibodies. E11 Ly-6A-GFP embryo slice stained with the indicated antibodies directly labeled with phycoerythrin (PE) (red) and Alexa Fluor 647 (blue). GFP signal (green). The merged image is shown with and without the transmitted light. Image orientation: ventral side of the embryo to the left. Scale bar: 10 m. Mice must be housed according to institutional guidelines and all animal procedures must be carried out in compliance with the standards for humane care and use of laboratory animals.

  2. 2.

    Supplementary Fig. 2

    Time-lapse confocal imaging and staining of the embryo slices. (a) The culture chamber is placed on the microscope heated stage and (b) covered with the lid of a 30 mm cm culture dish and with a transparent lid. (c) After removing the culture chamber from the microscope, remove the myeloid long-term medium from the top of the gel and place the gel with the help of a curved forceps on the lid of a 60 mm culture dish (the bottom of the gel is placed up). The antibody dilution is placed on the top of the gel. Mice must be housed according to institutional guidelines and all animal procedures must be carried out in compliance with the standards for humane care and use of laboratory animals.

Videos

  1. 1.

    Supplementary Movie 1

    Supplementary Movie 1 shows the embryo caudal part (separated from the dorsal tissue) stained with FITC anti-CD45 (green), PE anti-c-kit (red) and Alexa Fluor 647 anti-CD31 (blue). The video shows the sequential scanning images (merged of transmitted light and fluorescent channels) along the z-axis (40x lens).

  2. 2.

    Supplementary Movie 2

    Supplementary Movie 2 shows the closing of the aortic lumen during time-lapse confocal imaging of E10 embryo slice performed in medium (10x lens; merge of transmitted light and anti-CD31 staining (red)). Ventral side of the aorta upwards.

  3. 3.

    Supplementary Movie 3

    Supplementary Movie 3 shows the embryo dissection procedure.

  4. 4.

    Supplementary Movie 4

    Supplementary Movie 4 shows the intra-aortic injection procedure.

  5. 5.

    Supplementary Movie 5

    Supplementary Movie 5 shows the embryo slicing procedure.

About this article

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DOI

https://doi.org/10.1038/nprot.2011.401

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