The authors would like to acknowledge funding provided by the UCSF Resource Allocation Program.

All animal work was performed under strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Francisco.
1. Mouse Embryonic Lacrimal Glands (LG): Harvesting and Microdissection
<ol>
	<li>Following procedures approved by the Institutional Animals for Scientific Research Ethics committee, euthani...

<ol>
	<li>Liu, K. C., Huynh, K., Grubbs, J., Davis, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoimmunity+in+the+pathogenesis+and+treatment+of+keratoconjunctivitis+sicca.">Autoimmunity in the pathogenesis and treatment of keratoconjunctivitis sicca.</a> Current allergy and asthma reports. 14, 403 (2014).</li><li>Makarenkova, H. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF10+is+an+inducer+and+Pax6+a+competence+factor+for+lacrimal+gland+development.">FGF10 is an inducer and Pax6 a competence factor for lacrimal gland development.</a> Development. 127, 2563-2572 (2000).</li><li>Dartt, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+regulation+of+lacrimal+gland+secretory+processes:+relevance+in+dry+eye+diseases.">Neural regulation of lacrimal gland secretory processes: relevance in dry eye diseases.</a> Progress in retinal and eye research. 28, 155-177 (2009).</li><li>Nigam, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+can+the+intrinsic+power+of+branching+morphogenesis+be+used+for+engineering+epithelial+tissues+and+organs.">Concise review: can the intrinsic power of branching morphogenesis be used for engineering epithelial tissues and organs.</a> Stem cells translational medicine. 2, 993-1000 (2013).</li><li>Qu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosaminoglycan-dependent+restriction+of+FGF+diffusion+is+necessary+for+lacrimal+gland+development.">Glycosaminoglycan-dependent restriction of FGF diffusion is necessary for lacrimal gland development.</a> Development. 139, 2730-2739 (2012).</li><li>Rebustini, I. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MT2-MMP-dependent+release+of+collagen+IV+NC1+domains+regulates+submandibular+gland+branching+morphogenesis.">MT2-MMP-dependent release of collagen IV NC1 domains regulates submandibular gland branching morphogenesis.</a> Developmental cell. 17, 482-493 (2009).</li><li>Hoffman, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+Expression+Profiles+of+Mouse+Submandibular+Gland+Development:+FGFR1+Regulates+Branching+Morphogenesis+in+Vitro+Through+BMP-+and+FGF-Dependent+Mechanisms.">Gene Expression Profiles of Mouse Submandibular Gland Development: FGFR1 Regulates Branching Morphogenesis in Vitro Through BMP- and FGF-Dependent Mechanisms.</a> Development. 129, 24-5778 (2002).</li><li>Steinberg, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGFR2b+signaling+regulates+ex+vivo+submandibular+gland+epithelial+cell+proliferation+and+branching+morphogenesis.">FGFR2b signaling regulates ex vivo submandibular gland epithelial cell proliferation and branching morphogenesis.</a> Development. 132, 1223-1234 (2005).</li><li>Knox, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis. 329, 1645-1647 (2010).</li><li>Magenheim, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+restrain+pancreas+branching,+differentiation+and+growth.">Blood vessels restrain pancreas branching, differentiation and growth.</a> Development. 138, 4743-4752 (2011).</li><li>Jaskoll, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF10/FGFR2b+signaling+plays+essential+roles+during+in+vivo+embryonic+submandibular+salivary+gland+morphogenesis.">FGF10/FGFR2b signaling plays essential roles during in vivo embryonic submandibular salivary gland morphogenesis.</a> BMC developmental biology. 5, 11 (2005).</li><li>Takasato, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directing+human+embryonic+stem+cell+differentiation+towards+a+renal+lineage+generates+a+self-organizing+kidney.">Directing human embryonic stem cell differentiation towards a renal lineage generates a self-organizing kidney.</a> Nature cell biology. 16, 118-126 (2014).</li><li>Knox, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parasympathetic+stimulation+improves+epithelial+organ+regeneration.">Parasympathetic stimulation improves epithelial organ regeneration.</a> Nature communications. 4, 1494 (2013).</li></ol>

The versatility to culture and manipulate the LG ex vivo provides significant advantages for studying its development. This includes the speed at which the researcher is able to test hypotheses and the multitude of perturbations that can be performed to assess how epithelial, neuronal, endothelial and mesencyhmal cells interact to form the organ. However, there are a number of caveats when utilizing this model. First, by virtue of its isolation the gland is no longer connected to the peripheral vasculature or ne...

The lacrimal gland (LG) is responsible for aqueous tear secretion critical for visual acuity and the health, maintenance, and protection of the cells of the ocular surface. LG dysfunction results in one of the most common and debilitating ocular disorders: aqueous deficient Dry Eye Disease, which is characterized by ocular irritation, light sensitivity and decreased vision1. In the human the LG resides in the orbit above the lateral end of the eye where 3 - 5 excretory ducts deposit tears onto the ocular surface. The mouse has three pairs of major ocular glands, the most studied of which is the lacrimal gland (LG) located anterior and ventral to the ear (exorbital) with tears traveling to the eye via a single excretory duct. 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 (Figure 1)2. During development the epithelium becomes vascularized as well as heavily innervated by the parasympathetic nerves of the pterygopalatine ganglion and to a lesser extent by sympathetic nerves from the superior cervical ganglion3. Interactions between each of these cells types i.e. neuronal, epithelial, endothelial and mesenchymal cells, are essential to the function and maintenance of the adult tissue. However, the underlying molecular mechanisms coordinating LG development and regeneration as well as how inter-cell type communication guides these processes remains unclear.
The advent of embryonic ex vivo culture techniques has allowed the identification of developmental and regenerative pathways in multiple branching organs4. Culturing ex vivo gives the researcher the ability to manipulate the organ (mechanical, genetic or chemical) under defined conditions as well as to characterize organ development and cell-cell interactions in real time. The exorbital LG of the mouse is highly amenable to this technique and recent studies have defined signaling systems that regulate its development2,5. However, despite the need to understand molecular cues underpinning LG development and regeneration, it currently remains understudied, likely due to the technical difficulties in isolating the organ. In this paper, we describe how to isolate and perform ex vivo culture of the embryonic murine LG to define developmental programs.

<table border="0" cellpadding="0" cellspacing="0" width="1767">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Name</td>
			<td style="width:129px;">Catalog #.</td>
			<td style="width:213px;">Company</td>
			<td style="width:997px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM/F12 without HEPES</td>
			<td>SH3027101</td>
			<td>Thermo Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td>P0781</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Albumin solution from bovine serum</td>
			<td>A9576</td>
			<td>Sigma-Aldrich</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 16% Solution</td>
			<td>15710</td>
			<td>Electron Microscopy Sciences</td>
			<td>Diluted to 4% in 1X PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline 10X</td>
			<td>BP665-1</td>
			<td>Fisher Scientific</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffered Saline 1X</td>
			<td></td>
			<td></td>
			<td>Prepared from 10X stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">holo-Transferrin bovine</td>
			<td>T1283</td>
			<td>Sigma-Aldrich</td>
			<td>25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">L- Ascorbic acid (Vitamin C)</td>
			<td>A4544</td>
			<td>Sigma-Aldrich</td>
			<td>25 mg/ml stock solution in water. Freeze single-use aliquots at -20C. Add to DMEM/F12 media to a final concentration of 50 ug/mL.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BioLite 100mm Tissue Culture Treated Dishes</td>
			<td>130182</td>
			<td>Thermo Scientific</td>
			<td>Non-tissue culture-treated plates can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo Microscope with substage iluminator</td>
			<td>Stemi 2000</td>
			<td>Carl Zeiss</td>
			<td rowspan="2">Any stereo dissecting microscope can be used that has a transmitted light base.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Substage Illuminator Base for Stereo Microscope</td>
			<td>TLB 4000</td>
			<td>Diagnostics Instruments, Inc.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 35mm Tissue Culture Treated Dishes</td>
			<td>353001</td>
			<td>Corning</td>
			<td>Non-tissue culture-treated plates can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mm uncoated glass bottom dishes</td>
			<td>P50G-1.5-14-F</td>
			<td>MatTek Corporation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Widefield fluorescence microscope</td>
			<td>Axio Observer Z1</td>
			<td style="width:213px;">Carl Zeiss</td>
			<td>Any fluorescence microscope (upright, inverted or stereo dissecting microscope) can be used to monitor GFP expression at low magnification with an attached 
			digital camera.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>TCS SP5</td>
			<td style="width:213px;">Leica Microsystems</td>
			<td>Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td>Ideal for harvesting glands from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SWISS Micro-Fine Forceps, #5 (11.2cm)</td>
			<td>17-305X</td>
			<td>Integra LifeSciences Corporation</td>
			<td>Fine tips are required for removing mesenchyme from epithelium. Tungsten needles can also be used.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont Standard Tip Forceps, #5 (11cm)</td>
			<td>91150-20</td>
			<td>Fine Science Tools (USA), Inc.</td>
			<td>Ideal for harvesting glands from embryos</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reusable Plastic Surgical Knife Handles, style no. 3.</td>
			<td>4-30</td>
			<td colspan="2">Integra LifeSciences Corporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stainless Steel Sterile Surgical Blades, no. 10</td>
			<td>4-310</td>
			<td colspan="2">Integra LifeSciences Corporation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNAqueous-Micro Total RNA Isolation Kit</td>
			<td>AM1931</td>
			<td>Life Technologies</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cultrex 3D Culture Matrix Laminin I</td>
			<td>3446-005-01</td>
			<td>Trevigen</td>
			<td>6 mg/ml stock diluted 1:1 with DMEM/F12</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:428px;">Timed-pregnant Crl:CD1(ICR) mice</td>
			<td></td>
			<td style="width:213px;">Charles River Labs</td>
			<td>Embryos are harvested on day 14 (with day of plug discovery designated as day 0).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nucleopore Track-Etched Hydrophilic Membranes, 0.1 um pre size, 13mm</td>
			<td>110405</td>
			<td>Whatman</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Timed-pregnant Pax6-Cre,GFP mice</td>
			<td>Tg(Pax-Cre, GFP)1Pgr</td>
			<td>The Jackson Laboratory</td>
			<td>Optional mice for learning how to locate the LG.</td>
		</tr>
	</tbody>
</table>

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 LG develops through the process of epithelial branching morphogenesis. Brightfield images of embryonic LGs dissected at e14, e15, e16, e17, and P2 illustrate this event.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/51970/51970fig1highres.jpg" width="600" /> 
Figure 1. The LG develops through the process of epithelial branching morphogenesis. Brightfield images of embryonic LGs freshly dissected at e14, e15, e1...

Watch this Scientific Journal Video about Manipulating the Murine Lacrimal Gland at JoVE.com

Manipulating the Murine Lacrimal Gland

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 ...

Research

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Biology

20.6K vues.  University of California San Francisco.  La glande lacrymale (LG) est un organe de branchement qui produit les composants aqueux de larmes nécessaires pour le maintien de la vision et la santé oculaire. Nous décrivons ici murin LG dissection et ex vivo des techniques de culture de déchiffrer les voies impliquées dans le développement LG signalisation. 

Vidéo: Manipulating the Murine Lacrimal Gland

Silicon Microchips for Manipulating Cell-cell Interaction

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from development to malignant transformation.&nbsp; In particular, interaction with neighboring stroma has been implicated in a plethora of biological phenomena; however, conventional techniques limit the ability to interrogate the spatial and dynamic elements of such interactions.
In Micromechanical Reconfigurable Culture (RC), we employ a micromachined silicon substrate with moving parts to dynamically control cell-cell interactions through mechanical repositioning. Previously, this method has been applied to investigate intercellular communication in co-cultures of hepatocytes and non-parenchymal cells, demonstrating time-dependent interactions and a limited range for soluble signaling 1.
Here, we describe in detail the preparation and use of the RC system. We begin by demonstrating the handling of the device parts using tweezers, including actuating between the gap and contact configurations (cell populations separated by a narrow 80-&micro;m gap, or in direct intimate contact). Next, we detail the process of preparing the substrates for culture, and the multi-step cell seeding process required for obtaining confluent cell monolayers. Using live microscopy, we then illustrate real-time manipulation of cells between the different possible experimental configurations. Finally, we demonstrate the steps required in order to regenerate the device surface for reuse: toluene and piranha cleaning, polystyrene coating, and oxygen plasma treatment.

This article describes an experimental approach for dynamic regulation of cell-cell interactions between adherent cells on a micrometer scale. Manipulation of intercellular communication between hepatocytes and stromal cell is demonstrated. The developed platform enables investigation of cell-cell interactions in a variety of biological processes, including development and pathogenesis.

The role of the cellular microenvironment is recognized as crucial in determining cell fate and function in virtually all mammalian tissues from ...

Using Laser Tweezers For Manipulating Isolated Neurons In Vitro

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult retinal neurons in vitro. Procedures for preparing retinal cell cultures start with the dissection, digestion and trituration of the retina, and end with the plating of isolated retinal cells on dishes made especially for use with laser tweezers. These dishes are divided into a cell adhesive half and a cell repellant half. The cell adhesive side is coated with a layer of Sal-1 antibodies, which provide a substrate upon which our cells grow. Other adhesive substrates could be used for other cell types. The cell repellant side is coated with a thin layer of poly-HEMA. The cells plated on the poly-HEMA side of the dish are trapped with the laser tweezers, transported and then placed adjacent to a cell on the Sal-1 side to create a pair. Formation of cell groups of any size should be possible with this technique. "Laser-tweezers-controlled micromanipulation" means that the investigator can choose which cells to move, and the desired distance between the cells can be standardized. Because the laser beam goes through transparent surfaces of the culture dish, cell selection and placement are done in an enclosed, sterile environment. Cells can be monitored by video time-lapse and used with any cell biological technique required. This technique may help investigations of cell-cell interactions.

This video describes the manipulation of cultured neurons using laser tweezers in vitro.

In this paper and video, we describe the protocols used in our laboratory to study the targeting preferences of regenerating cell processes of adult ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

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 ...

Isolation of Murine Valve Endothelial Cells

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and surrounded by a monolayer of valve endothelial cells (VECs). VECs play essential roles in establishing the valve structures during embryonic development, and are important for maintaining life-long valve integrity and function. In contrast to a continuous endothelium over the surface of healthy valve leaflets, VEC disruption is commonly observed in malfunctioning valves and is associated with pathological processes that promote valve disease and dysfunction. Despite the clinical relevance, focused studies determining the contribution of VECs to development and disease processes are limited. The isolation of VECs from animal models would allow for cell-specific experimentation. VECs have been isolated from large animal adult models but due to their small population size, fragileness, and lack of specific markers, no reports of VEC isolations in embryos or adult small animal models have been reported. Here we describe a novel method that allows for the direct isolation of VECs from mice at embryonic and adult stages. Utilizing the Tie2-GFP reporter model that labels all endothelial cells with Green Fluorescent Protein (GFP), we have been successful in isolating GFP-positive (and negative) cells from the semilunar and atrioventricular valve regions using fluorescence activated cell sorting (FACS). Isolated GFP-positive VECs are enriched for endothelial markers, including CD31 and von Willebrand Factor (vWF), and retain endothelial cell expression when cultured; while, GFP-negative cells exhibit molecular profiles and cell shapes consistent with VIC phenotypes. The ability to isolate embryonic and adult murine VECs allows for previously unattainable molecular and functional studies to be carried out on a specific valve cell population, which will greatly improve our understanding of valve development and disease mechanisms.

The ability to isolate heart valve endothelial cells (VECs) is critical for understanding mechanisms of valve development, maintenance, and disease. Here we describe the isolation of VECs from embryonic and adult Tie2-GFP mice using FACS that will allow for studies determining the contribution of VECs in developmental and disease processes.

Normal valve structures consist of stratified layers of specialized extracellular matrix (ECM) interspersed with valve interstitial cells (VICs) and ...

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 Murine Coronary Vascular Smooth Muscle Cells

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs from the coronary circulation. Hearts with intact aortic arches were removed and perfused via retrograde Langendorff with digestion solution containing 300 Units/ml of collagenase type II, 0.1 mg/ml soybean trypsin inhibitor and 1 M CaCl2. The perfusates were collected at 15 min intervals for 90 min, pelleted by centrifugation, resuspended in plating media, and plated on tissue culture dishes. VSMCs were characterized by presence of SM22&#945;, &#945;-SMA, and vimentin. One of the main advantages of using this technique is the ability to isolate VSMCs from the coronary circulation of mice. Although the small number of cells obtained can limit some of the applications for which the cells can be utilized, isolated coronary VSMCs can be used in a variety of well-established cell culture techniques and assays. Studies investigating VSMCs from genetically modified mice can provide further information about structure-function and signaling processes associated with vascular pathologies.

The purpose of this protocol is to demonstrate the isolation and culture techniques of murine primary vascular smooth muscle cells (VSMCs) from the coronary circulation. Once VSMCs have been isolated, they can be used for many standard culture techniques.

While the isolation and culture of vascular smooth muscle cells (VSMCs) from large vessels is well established, we sought to isolate and culture VSMCs ...

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, ...

Isolation of Myoepithelial Cells from Adult Murine Lacrimal and Submandibular Glands

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ductal epithelial, and myoepithelial cells (MECs). MECs express alpha smooth muscle actin (&#945;SMA) and have a contractile function. They are found in multiple glandular organs and are of ectodermal origin. In addition, the LG contains SMA+ vascular smooth muscle cells of endodermal origin called pericytes: contractile cells that envelop the surface of vascular tubes. A new protocol allows us to isolate both MECs and pericytes from adult murine LGs and submandibular glands (SMGs). The protocol is based on the genetic labeling of MECs and pericytes using the SMACreErt2/+:Rosa26-TdTomatofl/fl mouse strain, followed by preparation of the LG single-cell suspension for fluorescence activated cell sorting (FACS). The protocol allows for the separation of these two cell populations of different origins based on the expression of the epithelial cell adhesion molecule (EpCAM) by MECs, whereas pericytes do not express EpCAM. Isolated cells could be used for cell cultivation or gene expression analysis.

The lacrimal gland (LG) has two cell types expressing &#945;-smooth muscle actin (&#945;SMA): myoepithelial cells (MECs) and pericytes. MECs are of ectodermal origin, found in many glandular tissues, while pericytes are vascular smooth muscle cells of endodermal origin. This protocol isolates MECs and pericytes from murine LGs.

The lacrimal gland (LG) is an exocrine tubuloacinar gland that secretes an aqueous layer of tear film. The LG epithelial tree is comprised of acinar, ...