Name: intravital subcellular microscopy of the mammary gland
Uploaded: 2022-06-24T14:00:00
Description: Watch this Scientific Journal Video about Intravital Subcellular Microscopy of the Mammary Gland at JoVE.com

The authors thank Sherry Rausch and Samri Gebre (National Cancer Institute, NIH) for animal management and care and James Mather for help in producing a range of plastic spacers.&#160;This research was supported [in part] by the Intramural Research Program of the NIH.

All animal procedures were approved by the Institutional Animal Care and Use Committee of the Center for Cancer Research, National Cancer Institute, the National Institutes of Health in compliance with the US National Research Council&#39;s Guide for the Care and Use of Laboratory Animals, the US Public Health Service&#39;s Policy on Humane Care and Use of Laboratory Animals, and the Guide for the Care and Use of Laboratory Animals. For this work, the number 4 glands of female primiparous mice (aged 4-5 months, day 10 of...

<ol>
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Whether to use a one- or multiphoton microscope depends upon the questions being asked, the nature and location of the tissue to be imaged, and the resolution required. Multiphoton microscopes are based on generating two or more low-energy photons in the near-infrared, which can penetrate tissues to a greater depth with less phototoxicity than one-photon microscopes29,30. In addition, the fluorophore is only excited at the focal point, which reduces light scatter...

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 secretory mechanisms during lactation8,9, and oxytocin-mediated contraction of myoepithelial cells9,10. Two main methods have been developed that take advantage of the gland&#39;s accessibility between the skin and body wall.
In the first approach, an acrylic or glass window is inserted into the skin and secured with a metal retaining ring1,3,11. The mice tolerate the surgery well, and various phenomena can be analyzed on an intermittent basis in the same animal over several weeks. This method has proved especially useful for lineage tracing1,12 and monitoring the invasion and progression of mammary tumors in situ3,11. However, resolution below the whole-cell level has proven difficult because the gland is still attached to the body wall and is thus subject to motion artifacts caused by respiration and heartbeat.
In the second approach, the gland is surgically exposed on a skin flap with intact vasculature and stabilized on the microscope stage with spacers4,9,13. A portion of the gland is thus effectively separated from the abdominal wall, and motion artifacts are minimized. In most cases, the exposed parenchyma is placed directly on the coverslip with the mouse ventral side down on an inverted microscope. In a recent modification, the mouse was placed supine on an upright microscope, and the exposed gland was protected in a fluid-filled cell sealed with a coverslip2. This latter configuration allows access to the parenchymal surface for experimental manipulation during imaging. Resolution down to &#60;1 &#181;m, in either case, permits analysis of intracellular processes, as exemplified by the tracking of lipid droplets (LDs) in mammary epithelial cells9.
The present protocol details a facile method for the intravital imaging of mammary epithelial cells at the sub-cellular level using the biogenesis, transport, and secretion of LDs during lactation as an example. This approach is widely applicable to many other processes, including the transport and secretion of milk proteins14, the transcytosis of proteins from the serosal side of the epithelium to the alveolar lumen15,16, and the remodeling of the gland during involution17,18.
Mice expressing a fluorescent protein are preferred for most intravital experiments to facilitate the selection of appropriate areas for imaging and as a morphological reference marker. A wide range of suitable transgenic and knock-in mice are available, which express fluorescent protein markers in cellular compartments, cytoskeletal elements, membranes, and organelles19. In the examples given, the EGFPcyto FvB mouse was used, in which enhanced green fluorescent protein (EGFP) is targeted to the cytoplasm in most cells20 (denoted EGFPcyto), and the C57BL/6J Tomato (mT/mG) mouse21, which is a double fluorescent Cre line encoding tdTomato and EGFP genes. EGFP expression is enabled through Cre-mediated excision of the tdTomato gene. Either fluorophore is targeted to the plasma membrane in most cells through a sequon derived from the MARCKS protein21. In this work, mice expressing the red tdTomato fluorophore are denoted tdTomatomembr (mT), and mice expressing EGFP, after excision of the tdTomato gene are denoted EGFPmembr (mG).
Mice have five pairs of mammary glands on either side of the ventral midline, three in the thoracic region (numbered 1-3) and two in the inguinal region (numbered 4-5) (Figure 1A). For ISMic, the inguinal glands are the most accessible and easiest to stabilize, as they are furthest away from global motions associated with respiration and heartbeat in the thorax.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>488 laser</td>
			<td>Melles-Griot</td>
			<td>-</td>
			<td>CW&#160; laser 50 mW</td>
		</tr>
		<tr>
			<td>60x PLAPON oil immersion objective (NA 1.42)</td>
			<td>Olympus</td>
			<td>1-U2B933</td>
			<td>Lens Confocal microscope</td>
		</tr>
		<tr>
			<td>633 laser</td>
			<td>Melles-Griot</td>
			<td>-</td>
			<td>CW He-Ne laser 12 mW</td>
		</tr>
		<tr>
			<td>63x objective (NA 1.40, HC PL APO CS2)</td>
			<td>Leica</td>
			<td>11506350</td>
			<td>Lens Two-photon microscope</td>
		</tr>
		<tr>
			<td>BA 410-460 nm</td>
			<td>Chroma</td>
			<td>-</td>
			<td>Band-pass filter</td>
		</tr>
		<tr>
			<td>BA 495-540 nm</td>
			<td>Chroma</td>
			<td>-</td>
			<td>Band-pass filter</td>
		</tr>
		<tr>
			<td>BA 505-605 nm</td>
			<td>Chroma</td>
			<td>-</td>
			<td>Band-pass filter</td>
		</tr>
		<tr>
			<td>BA 655-755 nm</td>
			<td>Chroma</td>
			<td>-</td>
			<td>Band-pass filter</td>
		</tr>
		<tr>
			<td>Boron-dipyrromethane (BODIPY) 665/676</td>
			<td>Thermo Fisher Scientific</td>
			<td>B3932</td>
			<td>Lipid peroxiation sensor</td>
		</tr>
		<tr>
			<td>Carbomer-940</td>
			<td>Snowdrift Farm</td>
			<td>739601480651</td>
			<td>Gel</td>
		</tr>
		<tr>
			<td>Catheter</td>
			<td>Terumo</td>
			<td>SV27EL</td>
			<td>Winged infusion sets&#160;</td>
		</tr>
		<tr>
			<td>Cauterizer&#160;</td>
			<td>Braintree Scientific, Inc</td>
			<td>GEM 5917</td>
			<td>Cautery system</td>
		</tr>
		<tr>
			<td>CMV-Cre mouse&#160;</td>
			<td>Jackson lab</td>
			<td>006054</td>
			<td>Mouse line</td>
		</tr>
		<tr>
			<td>Coverslip</td>
			<td>Bioptechs</td>
			<td>-</td>
			<td>30mm diameter coverlip for inverted microscope</td>
		</tr>
		<tr>
			<td>Curity 4x4 inch all purpose sponge gauze</td>
			<td>Covidien</td>
			<td>9024</td>
			<td>Sponge</td>
		</tr>
		<tr>
			<td>EGFPcyto mouse</td>
			<td>Jackson lab</td>
			<td>003291</td>
			<td>Mouse line</td>
		</tr>
		<tr>
			<td>Fiji/ImageJ software</td>
			<td>Open source</td>
			<td>-</td>
			<td>Free software tool</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Braintree Scientific, Inc</td>
			<td>FC003 8</td>
			<td>Tissue forceps</td>
		</tr>
		<tr>
			<td>Fluoview 1000 microscope</td>
			<td>Olympus</td>
			<td>FV1000</td>
			<td>Confocal microscope</td>
		</tr>
		<tr>
			<td>FluoView software</td>
			<td>Olympus</td>
			<td>-</td>
			<td>Confocal microscope and Two-photon microscope</td>
		</tr>
		<tr>
			<td>Hand-held electric razor</td>
			<td>Braintree Scientific, Inc</td>
			<td>CLP-8786-451A</td>
			<td>Cordless clipper</td>
		</tr>
		<tr>
			<td>Heat pad</td>
			<td>Braintree Scientific, Inc</td>
			<td>DPIP</td>
			<td>Heat pad for animals</td>
		</tr>
		<tr>
			<td>HyD detectors</td>
			<td>Leica</td>
			<td>-</td>
			<td>Leica 4Tune spectral detector</td>
		</tr>
		<tr>
			<td>Imaris software</td>
			<td>Bitplane / Oxford instruments</td>
			<td>-</td>
			<td>Commercial software tool</td>
		</tr>
		<tr>
			<td>Ingisht X3 tunable laser</td>
			<td>Spectra Physics</td>
			<td>Insight X3</td>
			<td>Tunable Pulse-Laser</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>VetOne</td>
			<td>13985-046-40</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>Ketamine&#160;</td>
			<td>VetOne</td>
			<td>13985-702-10</td>
			<td>Anesthetic</td>
		</tr>
		<tr>
			<td>LAS X Software</td>
			<td>Leica</td>
			<td>-</td>
			<td>Two-photon microscope software tool</td>
		</tr>
		<tr>
			<td>Mai-Tai tunable laser</td>
			<td>Spectra Physics</td>
			<td>Mai-Tai</td>
			<td>Laser</td>
		</tr>
		<tr>
			<td>MetaMorph</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>Commercial software tool</td>
		</tr>
		<tr>
			<td>Monodansylpentane AUTODOT</td>
			<td>Abcepta</td>
			<td>Sm1000a</td>
			<td>Lipid droplet dye</td>
		</tr>
		<tr>
			<td>MPE-RS microscope</td>
			<td>Olympus</td>
			<td>IX70</td>
			<td>Two-photon microscope</td>
		</tr>
		<tr>
			<td>mT/mG mouse</td>
			<td>Jackson lab</td>
			<td>007676</td>
			<td>Mouse line</td>
		</tr>
		<tr>
			<td>Objective heater</td>
			<td>Bioptechs</td>
			<td>150819</td>
			<td>Objective heater for both confocal and two-photon microscopes</td>
		</tr>
		<tr>
			<td>Oxygen-saturated respiration chamber</td>
			<td>Patterson Scientific</td>
			<td>78933385, SAS3 and EVAC4</td>
			<td>Gas Anesthesia and evacuation system&#160;</td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Heathrow Scientific</td>
			<td>HS234526B</td>
			<td>Semi-transparent, flexible, thermoplastic film</td>
		</tr>
		<tr>
			<td>PMT detector</td>
			<td>Olympus</td>
			<td>-</td>
			<td>Descanned&#160;&#160; detectors</td>
		</tr>
		<tr>
			<td>PMT detector</td>
			<td>LSM-Technology</td>
			<td>Custom built</td>
			<td>Non-Descanned Detectors</td>
		</tr>
		<tr>
			<td>Pump</td>
			<td>Harvard Apparatus</td>
			<td>703602, 704402</td>
			<td>Nanomite injector and controller</td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Quality Biological</td>
			<td>114-055-721EA</td>
			<td>Normal saline</td>
		</tr>
		<tr>
			<td>Sharp blunt-ended scissors</td>
			<td>Braintree Scientific, Inc</td>
			<td>SCT-S 508</td>
			<td>Surgical scissors</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Covidien</td>
			<td>22-257-150</td>
			<td>1mL tuberculin syringe</td>
		</tr>
		<tr>
			<td>TCS SP8 Dive Spectral microscope</td>
			<td>Leica</td>
			<td>SP8</td>
			<td>Two-photon microscope</td>
		</tr>
		<tr>
			<td>Tweezers&#160;</td>
			<td>Braintree Scientific, Inc</td>
			<td>FC032</td>
			<td>Tissue forceps</td>
		</tr>
		<tr>
			<td>Xylazine&#160;</td>
			<td>VetOne</td>
			<td>13985-704-10</td>
			<td>Anesthetic</td>
		</tr>
	</tbody>
</table>

intravital subcellular microscopy of the mammary gland

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&#160;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 &#8805; 0.7 &#181;m. Time-lapse images can be recorded by standard laser scanning confocal microscopy down to a depth of 15-25 &#181;m 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.

Milk is secreted from polarized alveolar epithelial cells, which differentiate during pregnancy from the buds of an extensive ductular tree26 (Figure 2A). Precursors for milk synthesis are assimilated across basal/lateral membranes and completed products are secreted across the apical surface into a central &#34;milk space&#34;. The basal side of each alveolus is covered by a stellate array of myoepithelial cells (Figure 2A), which are pr...

Watch this Scientific Journal Video about Intravital Subcellular Microscopy of the Mammary Gland at JoVE.com

Intravital Subcellular Microscopy of the Mammary Gland

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

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

To begin, place the shaved, anesthetized mouse on a warming pad. Pinch the skin with tweezers near the fourth nipple and cut the raised skin with sharp scissors to make a midline incision of approximately one centimeter. Make circular incisions around the gland in both cranial and caudal directions, and carefully peel the skin away from the abdomen.Remove any exogenous blood promptly with physiological saline to avoid subsequent loss of optical resolution. To minimize blood loss, seal any prominent blood vessels with a handheld cauterizer. Then gently tease the surface layer with fine forceps to remove superficial connective and adipose tissue.Keep the exposed gland moist with physiological saline. If required, treat the gland with exogenous agents, for example, pharmacological agents or organelle specific dyes by bathing the exposed glands during surgery. Protect the abdominal wall with a semi-transparent, flexible thermoplastic film.Carefully position the mouse with the abdominal side down on an inverted microscope stage such that the skin flap extends onto the central cover glass. Protect the exposed areas with a thin layer of gel. Tape the rear leg and tail to the stage.Then place a custom made notched spacer prepared from three taped cotton sticks between the skin flap and the body wall to cushion the gland from motion artifacts caused by breathing and heartbeat. Securely tape a plastic cover at either end of the stage opening to prevent the skin flap from sliding during the imaging. Insert a subcutaneous indwelling catheter under the dorsal skin attached to a tube, syringe, and pump.Using conventional fluorescence microscopy, confirm that the skin flap is stable with adequate blood flow. Perform conventional confocal microscopy of the EGFP cyto or EGFP membrane mouse labeled with BODIPY 665676 and two photon microscopy of the EGFP membrane mouse labeled with monodansylpentane and tdTomato membrane mouse labeled with monodansylpentane. In EGFP cyto mice, the cytoplasm of secretory epithelial cells was distinctly labeled by EGFP.At the same time, lipid droplets stained with BODIPY 665676 appeared as round fluorescent bodies, especially towards the apical surface. Time-lapse analysis showed BODIPY 665676 stained lipid droplets moving from basal to apical regions at variable speeds and fusing with each other in transit. In EGFP membrane mice, EGFP highlighted the plasma membranes of capillary and secretory epithelial cells and BODIPY stained lipid droplets were visible throughout the cytoplasm.Two photon microscopy of the mammary glands of either EGFP membrane or tdTomato membrane mice allowed a more comprehensive survey of gland morphology. Collagen fibers at the surface are visualized in the monodansylpentane channel through second harmonic generation. Further into the parenchyma, the secretory epithelium is revealed with basal, lateral, and apical plasma membranes and monodansylpentane stained lipid droplets.The central lumen becomes obvious further into the alveolus. The red tdTomato fluorophore was especially useful for labeling the capillary endothelium, unlike EGFP in the EGFP cyto mice.

intravital-subcellular-microscopy-of-the-mammary-gland

Research

JoVE Journal

Biology

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

Changes in Mammary Gland Morphology and Breast Cancer Risk in Rats

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland undergoes extensive modeling and re-modeling, alter susceptibility to carcinogen-induced mammary tumors. Similar findings have been described in humans: for example, high birthweight increases later risk of developing breast cancer, and dietary intake of soy during childhood decreases breast cancer risk. It is thought that these prenatal and postnatal dietary modifications induce persistent morphological changes in the mammary gland that in turn modify breast cancer risk later in life. These morphological changes likely reflect epigenetic modifications, such as changes in DNA methylation, histones and miRNA expression that then affect gene transcription . In this article we describe how changes in mammary gland morphology can predict mammary cancer risk in rats. Our protocol specifically describes how to dissect and remove the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology according to three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use the data to predict risk of developing mammary cancer.

Our protocol describes how to dissect the rat abdominal mammary gland and how to prepare mammary gland whole mounts. It also describes how to analyze mammary gland morphology using three end-points (number of terminal end buds, epithelial elongation and differentiation) and to use these results to predict mammary cancer risk in rats which were exposed to dietary modifications in utero or during prepuberty.

Studies in rodent models of breast cancer show that exposures to dietary/hormonal factors during the in utero and pubertal periods, when the mammary gland ...

Evaluation of Mammary Gland Development and Function in Mouse Models

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves composed of a number of terminal duct lobular units made of secretory alveoli and converging ducts1. In mice, a similar architecture is observed at pregnancy in which ducts and alveoli are interspersed within the connective tissue stroma. The mouse mammary gland epithelium is a tree like system of ducts composed of two layers of cells, an inner layer of luminal cells surrounded by an outer layer of myoepithelial cells denoted by the confines of a basement membrane2. At birth, only a rudimental ductal tree is present, composed of a primary duct and 15-20 branches. Branch elongation and amplification start at the beginning of puberty, around 4 weeks old, under the influence of hormones3,4,5. At 10 weeks, most of the stroma is invaded by a complex system of ducts that will undergo cycles of branching and regression in each estrous cycle until pregnancy2. At the onset of pregnancy, a second phase of development begins, with the proliferation and differentiation of the epithelium to form grape-shaped milk secretory structures called alveoli6,7. Following parturition and throughout lactation, milk is produced by luminal secretory cells and stored within the lumen of alveoli. Oxytocin release, stimulated by a neural reflex induced by suckling of pups, induces synchronized contractions of the myoepithelial cells around the alveoli and along the ducts, allowing milk to be transported through the ducts to the nipple where it becomes available to the pups 8. Mammary gland development, differentiation and function are tightly orchestrated and require, not only interactions between the stroma and the epithelium, but also between myoepithelial and luminal cells within the epithelium9,10,11. Thereby, mutations in many genes implicated in these interactions may impair either ductal elongation during puberty or alveoli formation during early pregnancy, differentiation during late pregnancy and secretory activation leading to lactation12,13. In this article, we describe how to dissect mouse mammary glands and assess their development using whole mounts. We also demonstrate how to evaluate myoepithelial contractions and milk ejection using an ex-vivo oxytocin-based functional assay. The effect of a gene mutation on mammary gland development and function can thus be determined in situ by performing these two techniques in mutant and wild-type control mice.

This method describes how to dissect and assess mammary gland development and function from mice. Excised mammary glands are assessed for the degree of development using whole mount while milk ejection is evaluated using an oxytocin-based myoepithelial cell contraction assay.

The human mammary gland is composed of 15-20 lobes that secrete milk into a branching duct system opening at the nipple. Those lobes are themselves ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of genes within the mammary epithelium has been difficult to achieve due to the lack of appropriate targeting molecules that are independent of developmental stages such as pregnancy and lactation. Herein, we describe a technique for localized delivery of reagents to the mammary gland at any stage in adulthood via intraductal injection into the nipples of mice. The injections can be performed on live mice, under anesthesia, and allow for a non-invasive and localized drug delivery to the mammary gland. Furthermore, the injections can be repeated over several months without damaging the nipple. Vital dyes such as Evans Blue are very helpful to learn the technique. Upon intraductal injection of the blue dye, the entire ductal tree becomes visible to the eye. Furthermore, fluorescently labeled reagents also allow for visualization and distribution within the mammary gland. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents, and small molecules.

A protocol for the non-invasive intraductal delivery of aqueous reagents to the mouse mammary gland is described. The method takes advantage of localized injection into the nipples of mammary glands targeting mammary ducts specifically. This technique is adaptable for a variety of compounds including siRNA, chemotherapeutic agents and small molecules.

Herein we describe a protocol to deliver various reagents to the mouse mammary gland via intraductal injections. Localized drug delivery and knock-down of ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Bovine Mammary Gland Biopsy Techniques

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, signaling pathways, and protein translation. This article describes two techniques for biopsy of the bovine mammary gland (MG). Three healthy Holstein dairy cows were the subjects. Before biopsies, cows were milked and subsequently restrained in a cattle chute. An analgesic (flunixin meglumine, 1.1 to 2.2 mg/kg of body weight) was administered via jugular intravenous [IV] injection 15-20 min prior to biopsy. For standing sedation, xylazine hydrochloride (0.01-0.05 mg/kg of body weight) was injected via the coccygeal vessels 5-10 min before the procedure. Once adequately sedated, the biopsy site was aseptically prepared and locally anaesthetized with 6 mL of 2% lidocaine hydrochloride via subcutaneous injection. Using aseptic technique, a 2 to 3 cm vertical incision was made using a number 10 scalpel. Core and needle biopsy tools were used. The core biopsy tool was attached to a cordless drill and inserted into the MG tissue through the incision using a clock-wise drill action. The needle biopsy tool was manually inserted into the incision site. Immediately after the procedure, an assistant applied pressure on the incision site for 20 to 25 min using a sterile towel to achieve hemostasis. Stainless steel surgical staples were used to oppose the skin incision. The staples were removed 10 days post-procedure. The main advantages of core and needle biopsies is that both approaches are minimally invasive procedures that can be safely performed in healthy cows. Milk yield following the biopsy was unaffected. These procedures require a short recovery time and result in fewer risks of complications. Specific limitations may include bleeding after the biopsy and infection on the biopsy site. Applications of these techniques include tissue collection for clinical diagnosis and research purposes, such as primary cell culture.

This article presents a bovine mammary gland biopsy using core and needle biopsy tools. Harvested tissue can be used for cell culture or to assess mammary physiology and metabolism including gene expression, protein expression, protein modifications, immunohistochemistry, and metabolite concentrations.

Bovine mammary gland biopsies allow researchers to collect tissue samples to study cell biology including gene expression, histological analysis, ...

Super-Resolution Live Cell Imaging of Subcellular Structures

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally been restricted to be used only on fixed samples or live cells outside of tissue labeled with strong fluorescent signal. Current super-resolution live cell imaging techniques require the use of special fluorescence probes, high illumination, multiple image acquisitions with post-acquisition processing, or often a combination of these processes. These prerequisites significantly limit the biological samples and contexts that this technique can be applied to.
Here we describe a method to perform super-resolution (~140 nm XY-resolution) time-lapse fluorescence live cell imaging in situ. This technique is also compatible with low fluorescent intensity, for example, EGFP or mCherry endogenously tagged at lowly expressed genes. As a proof-of-principle, we have used this method to visualize multiple subcellular structures in the Drosophila testis. During tissue preparation, both the cellular structure and tissue morphology are maintained within the dissected testis. Here, we use this technique to image microtubule dynamics, the interactions between microtubules and the nuclear membrane, as well as the attachment of microtubules to centromeres.
This technique requires special procedures in sample preparation, sample mounting and immobilizing of specimens. Additionally, the specimens must be maintained for several hours after dissection without compromising cellular function and activity. While we have optimized the conditions for live super-resolution imaging specifically in Drosophila male germline stem cells (GSCs) and progenitor germ cells in dissected testis tissue, this technique is broadly applicable to a variety of different cell types. The ability to observe cells under their physiological conditions without sacrificing either spatial or temporal resolution will serve as an invaluable tool to researchers seeking to address crucial questions in cell biology.

Presented here is a protocol for super-resolution live-cell imaging in intact tissue. We have standardized the conditions for imaging a highly sensitive adult stem cell population in its native tissue environment. This technique involves balancing temporal and spatial resolution to allow for the direct observation of biological phenomena in live tissue.

There has long been a crucial tradeoff between spatial and temporal resolution in imaging. Imaging beyond the diffraction limit of light has traditionally ...