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.

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JoVE Journal

Biology

788 次观看.  National Institutes of Health. 首先，将剃光的麻醉鼠标放在加热垫上。用镊子捏住第四个附近的皮肤，然后用锋利的剪刀剪开凸起的皮肤，做一个大约一厘米的中线切口。在腺体周围沿颅部和尾部方向做圆形切口，并小心地将皮肤从腹部剥离。立即用生理盐水去除任何外源性血液，以避免随后失去光学分辨率。为了尽量减少失血，请用手持式烧灼机密封任何突出的血管。然...