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A Whole-mount Immunofluorescence Protocol for Three-dimensional Imaging of the Embryonic Mammary Primordium

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Abstract

Whole-mount immunofluorescent staining facilitates the profiling of protein expression patterns within diverse and complex tissues. Thanks to the application of antibodies on whole mounted instead of sectioned specimens, this technique has many advantages with respect to the preservation of biological and pathological features of specimens when compared to conventional immunohistological methods. Here, we describe a protocol and optimal conditions of whole-mount immunofluorescence for studying the formation of mammary primordia. We also show an example three-dimensional reconstruction of a mammary primordium based on z-stacked images of a whole-mount stained specimen using confocal microscopy and image analysis software.

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Abbreviations

AR:

Androgen Receptor

DAPI:

4,6-diamidino-2-phenylindole dihydrochloride

DMF:

Dimethylformamide

E:

Embryonic day

EpCAM:

Epithelial cell adhesion molecule

ERα:

Estrogen receptor alpha

ME:

Mammary epithelium

MM:

Mammary mesenchyme

PB buffer:

Permeabilizing-blocking buffer

PBS:

Phosphate-buffered saline

PFA:

Paraformaldehyde

s-SHIP:

a shorter isoform of an SH2-containing inositol 5V-phosphatase

TBS:

Tris-buffered saline

References

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Acknowledgements

This work is funded by Breakthrough Breast Cancer, UK.

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Authors and Affiliations

  1. Division of Breast Cancer Research, Breakthrough Breast Cancer Research Centre, The Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB, UK

    Naoko Kogata & Beatrice A. Howard

Authors
  1. Naoko Kogata
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  2. Beatrice A. Howard
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Corresponding author

Correspondence to Beatrice A. Howard.

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Kogata, N., Howard, B.A. A Whole-mount Immunofluorescence Protocol for Three-dimensional Imaging of the Embryonic Mammary Primordium. J Mammary Gland Biol Neoplasia 18, 227–231 (2013). https://doi.org/10.1007/s10911-013-9285-5

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  • DOI: https://doi.org/10.1007/s10911-013-9285-5

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