Intravital Subcellular Microscopy of the Mammary Gland

Yeap Ng; Andrius Masedunskas; Marco Heydecker; Seham Ebrahim; Roberto Weigert; Ian H. Mather

doi:10.3791/63674

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Method Article

Intravital Subcellular Microscopy of the Mammary Gland

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DOI:

10.3791/63674

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June 24th, 2022

Yeap Ng¹ , Andrius Masedunskas¹^,² , Marco Heydecker¹ , Seham Ebrahim¹^,³ , Roberto Weigert¹ , Ian H. Mather¹

¹Laboratory of Cellular and Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, ²Charles Perkins Centre, Central Clinical School, Faculty of Medicine and Health, The University of Sydney, ³Department of Molecular Physiology and Biological Physics, School of Medicine, University of Virginia

Summary

The present protocol describes a facile technique for the intravital imaging of the lactating mouse mammary gland by laser scanning confocal and multiphoton microscopy.

Abstract

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory mechanisms. During pregnancy, the gland expands from a primitive ductal tree embedded in a fat pad to a highly branched alveolar network primed for the formation and secretion of colostrum and milk. Post-partum, the gland supplies all the nutrients required for neonatal survival, including membrane-coated lipid droplets (LDs), proteins, carbohydrates, ions, and water. Various milk components, including lactose, casein micelles, and skim-milk proteins, are synthesized within the alveolar cells and secreted from vesicles by exocytosis at the apical surface. LDs are transported from sites of synthesis in the rough endoplasmic reticulum to the cell apex, coated with cellular membranes, and secreted by a unique apocrine mechanism. Other preformed constituents, including antibodies and hormones, are transported from the serosal side of the epithelium into milk by transcytosis. These processes are amenable to intravital microscopy because the mammary gland is a skin gland and, therefore, directly accessible to experimental manipulation. In this paper, a facile procedure is described to investigate the kinetics of LD secretion in situ, in real-time, in live anesthetized mice. Boron-dipyrromethene (BODIPY)_665/676 or monodansylpentane are used to label the neutral lipid fraction of transgenic mice, which either express soluble EGFP (enhanced green fluorescent protein) in the cytoplasm, or a membrane-targeted peptide fused to either EGFP or tdTomato. The membrane-tagged fusion proteins serve as markers of cell surfaces, and the lipid dyes resolve LDs ≥ 0.7 µm. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 µm or by multiphoton microscopy for imaging deeper in the tissue. The mammary gland may be bathed with pharmacological agents or fluorescent dyes throughout the surgery, providing a platform for acute experimental manipulations as required.

Introduction

Intravital microscopy of the mouse mammary gland is attracting increased attention as a powerful method for analyzing a whole range of biological phenomena, including the origin and differentiation of stem cells¹^,², the progression of metastatic tumors³^,⁴^,⁵, and the role of ductal macrophages throughout mammary development and involution⁶. Through the development of Intravital Subcellular Microscopy (ISMic)⁷, investigations have been extended to membrane traffic and secr....