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 lactation) were surgically prepared in the right-hand supine position (Figure 1A).
1. Animal preparation
<ol>
	<li>Weigh the mouse on a top-loading balance to calculate the amount of anesthetic required.</li>
	<li>During and after applying anesthetics, maintain body temperature by placing the mouse on a warming pad.</li>
	<li>Anesthetize the mouse by brief exposure to isoflurane (3.0%-3.5%) in an oxygen-saturated respiration chamber (see Table of Materials) for 1-2 min, followed by an intraperitoneal injection of a mixture of 100 mg/kg of ketamine and 10 mg/kg of xylazine.</li>
	<li>Maintain animals under a deep plane of anesthesia during both surgery and imaging by further applying titrated doses of xylazine and ketamine (half to a quarter of the initial dose outlined in step 1.3) through an indwelling catheter (see step 3.4) every 45 min or so, as required. 
	NOTE: An optimal plane of anesthesia is most easily maintained by checking for palpebral and toe pinch reflexes every 10-20 min. The temperature of conscious mice is 36.5-38 &#176;C, which drops to 34.5 &#176;C under anesthesia.</li>
	<li>Cross-foster the pups22 if donor mice at a similar stage of lactation are available; otherwise, euthanize the litter. 
	&#8203;NOTE: Pups &#60;10 days of age should be euthanized by exposure to 3.0% isoflurane followed by decapitation. Pups &#8805;10 days of age should be euthanized via CO2 inhalation followed by cervical dislocation.</li>
</ol>
2. Surgery procedure
<ol>
	<li>Keep the mouse warm throughout surgery on a warming pad. Shave the fur from the skin surrounding the number 4 and 5 mammary glands (supine, right side) using a hand-held electric razor. Clean the shaved area with three alternating betadine and alcohol scrubs.</li>
	<li>Wipe all surgical instruments with 70% alcohol. Make a mid-line incision of ~1 cm close to the fourth nipple by pinching the skin with tweezers and cutting the raised skin with sharp scissors. Make circular incisions around the gland in both cranial and caudal directions and carefully peel the skin away from the abdomen. Keep the exposed skin moist with physiological saline.</li>
	<li>To minimize blood loss, seal any prominent blood vessels with a hand-held cauterizer (see Table of Materials) before cutting through them. Any exogenous blood should be promptly removed with physiological saline to avoid subsequent loss of optical resolution.</li>
	<li>Remove superficial connective and adipose tissue by gently teasing the surface layer with fine forceps.</li>
	<li>Keep the exposed gland moist with physiological saline and protect the abdominal wall with gel and semi-transparent, flexible, thermoplastic film (see Table of Materials). 
	NOTE: Steps 2.2-2.5 create a flap of skin with a portion of the gland and associated vasculature separated from the abdominal wall (Figure 1Bi-iv).</li>
	<li>If required, treat the gland with exogenous agents, e.g., pharmacological agents or organelle-specific dyes, by bathing the exposed gland during surgery. 
	&#8203;NOTE: In the examples given, LDs in either an EGFPcyto or EGFPmembr (mG) mouse were labeled with 1.0 mL of boron-dipyrromethene (BODIPY)665/676 (10 mM stock solution in dimethyl sulphoxide diluted 1 to a 1,000-fold in saline and applied for &#60;1 h) and in the tdTomatomembr (mT) or EGFPmembr (mG) mouse, with 0.5 mL of monodansylpentane23 (0.1M stock solution in dimethyl sulphoxide diluted 1 to a 1,000-fold in saline and applied for 5 min).</li>
</ol>
3. Imaging preparation
<ol>
	<li>Carefully position the mouse with the abdominal side down on a heated (37 &#176;C) inverted microscope stage, such that the skin flap extends onto the central cover glass (30 mm) (Figure 1Biv). Protect the exposed areas with a thin layer of gel.</li>
	<li>Stabilize the skin flap with custom-made spacers to cushion the gland from motion artifacts caused by breathing and heartbeat. To achieve this, place a notched spacer made from three taped cotton sticks (Figure 1Ci) between the skin flap and the body wall and tape the rear leg and tail to the stage behind the spacer (Figure 1Bv).</li>
	<li>Prevent the skin flap from sliding during imaging by securely taping a plastic cover (Figure 1Cii) at either end on the stage opening (Figure 1Bv).</li>
	<li>Insert a subcutaneous indwelling catheter under the dorsal skin attached to a tube, syringe, and pump (see Table of Materials).</li>
	<li>Confirm that the skin flap is stable with adequate blood flow by conventional fluorescence microscopy (Figure 1Bvi).</li>
	<li>Cover the mouse with sponge gauze (see Table of Materials) to keep warm during imaging, 
	&#8203;NOTE: Steps 3.1-3.5 are the most critical in ensuring the acquisition of high-resolution videos suitable for quantitative analysis. There is no point in proceeding beyond this stage unless tissue stability has been confirmed.</li>
</ol>
4. Microscopy
<ol>
	<li>For conventional confocal microscopy of the EGFPcyto or EGFPmembr (mG) mouse labeled with BODIPY665/676 (Figure 2, Video 1, and Video 2), use an inverted microscope equipped with a preheated 60x oil immersion objective, maintained at 37 &#176;C (see Table of Materials).
	<ol>
		<li>Separately detect EGFP and BODIPY665/676 using 488 and 633 lasers, respectively; for EGFP, excitation 488 nm, and emission 560 nm, with band-pass filter BA 505-605; for BODIPY665/676, excitation 633 nm and emission 668 nm, with band-pass filter BA 655-755.</li>
		<li>Collect images by line scan at either 4 or 8 &#181;s/pixel (512 x 512 pixels; 12 bits per pixel) every 5 s or 10 s for 1-2 h and store as TIFF files. Manually maintain scanned areas in-frame by correcting for x/y drift throughout the imaging cycle. Construct 3-D images from z-scans using software associated with the microscope (see Table of Materials).</li>
	</ol>
	</li>
	<li>For two-photon microscopy of the EGFPmembr (mG) mouse labeled with monodansylpentane (Figure 3, Video 3), use an inverted microscope equipped with a tunable laser and a 37 &#176;C preheated 30x objective (see Table of Materials).
	<ol>
		<li>Excite the gland at 910 nm and detect monodansylpentane and EGFP with two GaAs detectors, using 410-460 nm and 495-540 nm band-pass filters for blue and green emissions, respectively.</li>
		<li>Collect images by line scan at 2 &#181;s/pixel (320 x 320 pixels; 16 bits per pixel) and store them as OIR files. Manually maintain scanned areas in-frame by correcting x/y drift throughout the imaging cycle. Construct 3-D images from z-scans using software associated with the microscope.</li>
	</ol>
	</li>
	<li>For two-photon microscopy of the tdTomatomembr (mT) mouse labeled with monodansylpentane (Figure 4, Video 4), use an inverted microscope with tunable lasers, and a 37 &#176;C preheated 63x objective (see Table of Materials).
	<ol>
		<li>Excite specimens at 800 nm and 1,060 nm simultaneously and detect monodansylpentane with a HyD hybrid detector set at emission 418-471 nm and TdTomato with another HyD hybrid detector set at emission 595-667 nm.</li>
		<li>Collect the images by line scan with four lines averaging at 200 Hz (512 x 512 pixels; 12 bits per pixel) and store them as LIF files. Manually maintain scanned areas in-frame by correcting x/y drift throughout the imaging cycle. Construct 3-D images from z scans using software associated with the microscope.</li>
	</ol>
	</li>
</ol>
5. Euthanasia
<ol>
	<li>Euthanize the mice at the end of imaging via CO2 inhalation following institutionally approved protocols.</li>
</ol>
6. Creation of real-time videos
<ol>
	<li>Convert time sequences into TIFF files, and then into videos using appropriate software (see Table of Materials). 
	NOTE: Quantitative analysis of specific videos is beyond the scope of this paper and will require specific methods for the solution of specific problems. For example, see Ebrahim et al.24 for the role of actinomyosin complexes in exocytosis of saliva from the salivary gland, Meyer et al.25 for the dynamics of bile secretion from the liver, and Masedunskas et al.9 for analysis of LD secretion from mammary epithelial cells.</li>
</ol>

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None of the authors have any conflicting interests to declare.

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.

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

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

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Biology

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

Video: Intravital Subcellular Microscopy of the Mammary Gland

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 ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

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.