Intravital Subcellular Microscopy of the Mammary Gland

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

doi:10.3791/63674

Microscopía subcelular intravital de la glándula mamaria

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Biology
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JoVE Journal Biology

Intravital Subcellular Microscopy of the Mammary Gland

Microscopía subcelular intravital de la glándula mamaria

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04:14 min

June 24, 2022

DOI: 10.3791/63674-v

Yeap Ng¹, Andrius Masedunskas^1,2, Marco Heydecker¹, Seham Ebrahim^1,3, 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

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Overview

This protocol presents a technique for intravital imaging of the lactating mouse mammary gland utilizing laser scanning confocal and multiphoton microscopy. The study emphasizes the importance of maintaining optical resolution while minimizing blood loss during surgery.

Key Study Components

Research Area

Microscopy
Imaging techniques

Background

Intravital imaging provides insights into gland morphology and cellular dynamics.
Understanding the lactating mammary gland is crucial for developmental biology and cell biology.

Methods Used

Laser scanning confocal and multiphoton microscopy
Lactating mouse mammary gland
Use of EGFP and BODIPY dyes

Main Results

Distinct labeling of secretory epithelial cells and lipid droplets.
Time-lapse analysis showed dynamic movement of lipid droplets.
Comprehensive survey of gland morphology using dual fluorescence.

Conclusions

The study demonstrates a method to visualize complex biological processes in real-time.
This approach is relevant for further understanding mammary gland physiology and disease.

Frequently Asked Questions

What is the purpose of using laser scanning confocal microscopy?

It allows for high-resolution imaging of cellular structures in the mammary gland.

How does the protocol minimize blood loss?

By using a cauterizer on prominent blood vessels and saline to manage exogenous blood.

What dyes are used for labeling in this protocol?

EGFP and BODIPY are used for labeling secretory cells and lipid droplets.

Why is it important to maintain optical resolution?

High optical resolution is crucial for accurate imaging of cellular components and dynamics.

Can this method be applied to other tissues?

While this protocol is specific for the mammary gland, similar techniques could be adapted for other tissues.

What insights does time-lapse imaging provide?

It allows observation of the dynamics of lipid droplet movements within cells.

El presente protocolo describe una técnica sencilla para la obtención de imágenes intravitales de la glándula mamaria de ratón lactante mediante microscopía confocal y multifotónica de barrido láser.

Para comenzar, coloque el ratón afeitado y anestesiado sobre una almohadilla térmica. Pellizque la piel con pinzas cerca del cuarto pezón y corte la piel levantada con unas tijeras afiladas para hacer una incisión en la línea media de aproximadamente un centímetro. Haga incisiones circulares alrededor de la glándula en dirección craneal y caudal, y retire con cuidado la piel del abdomen.

Eliminar rápidamente la sangre exógena con solución salina fisiológica para evitar la pérdida posterior de la resolución óptica. Para minimizar la pérdida de sangre, selle los vasos sanguíneos prominentes con un cauterizador manual. A continuación, cepille suavemente la capa superficial con pinzas finas para eliminar el tejido conectivo y adiposo superficial.

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