Name: isolation of intact, whole mouse mammary glands for analysis of extracellular matrix expression and gland morphology
Uploaded: 2017-10-30T15:00:00
Description: Watch this Scientific Journal Video about Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology at JoVE.com

Forfatterne vil gerne anerkende April Wiles og Dr. Roger Broderson for hjælp til dyr obduktion og kirtel isolation, henholdsvis. Finansiering af dette arbejde blev støttet af Philadelphia College af osteopatisk medicin centre for kroniske lidelser af aldring.

procedurer, der involverer dyr emner i denne protokol blev gennemgået og godkendt af institutionelle Animal Care og brug Udvalget af Philadelphia College af osteopatisk medicin og alle teknikker blev gennemført under strenge etiske retningslinjer.
 1. prøven indkøb og forarbejdning <ol><li>Vælg passende animalske emne og sted i en CO 2 kammer. For dette eksperiment, bruge 8-10 ugers gammel kvinde har født B6. CG-Cav1tmMIs/J og C57BI/6J.</li>
	<li>Tænd gasflow til 30-40%. N...

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R., 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=Matrix+crosslinking+forces+tumor+progression+by+enhancing+integrin+signaling.">Matrix crosslinking forces tumor progression by enhancing integrin signaling.</a> Cell. 139 (5), 891-906 (2009).</li><li>Wang, J. 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K., 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=An+absence+of+stromal+caveolin-1+expression+predicts+early+tumor+recurrence+and+poor+clinical+outcome+in+human+breast+cancers.">An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor clinical outcome in human breast cancers.</a> Am J Pathol. 174 (6), 2023-2034 (2009).</li><li>Macias, H., Hinck, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+gland+development.">Mammary gland development.</a> Wiley Interdiscip Rev Dev Biol. 1 (4), 533-557 (2012).</li><li>Caligioni, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+reproductive+status/stages+in+mice.">Assessing reproductive status/stages in mice.</a> Curr Protoc Neurosci. , (2009).</li><li>Cannata, D., 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=Elevated+circulating+IGF-I+promotes+mammary+gland+development+and+proliferation.">Elevated circulating IGF-I promotes mammary gland development and proliferation.</a> Endocrinology. 151 (12), 5751-5761 (2010).</li><li>Thompson, C., Rahim, S., Arnold, J., Hielscher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+caveolin-1+alters+extracellular+matrix+protein+expression+and+ductal+architecture+in+murine+mammary+glands.">Loss of caveolin-1 alters extracellular matrix protein expression and ductal architecture in murine mammary glands.</a> PLoS One. 12 (2), 0172067 (2017).</li><li>Williams, C. 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I papiret, har vi beskrevet en teknik til at isolere intakt mus mælkekirtlerne downstream histologiske analyser af ECM udtryk og duktalt morfologi. Med hensyn til analyser af duktalt morfologi, giver denne metode mulighed for hurtig undersøgelse af duktalt arkitektur baseret ud af farvede histologiske sektioner. Andre metoder af duktalt analyser stole på injektioner af farvestoffer til aktiverer visualisering af duktalt træet, metoder, som kan være teknisk udfordrende og tidskrævende.
I ...

Brystkræft er karakteriseret ved en betydelig grad af væv fibrose1,2,3,4. Omtales som ECM, denne ikke-cellulære enhed findes i varierende grader i alle væv og består primært af en kompleks meshwork af fibrillar og ikke-fibrillar collagens, elastin, og glykoproteiner ud over forskellige signaling molekyler, der er afsondret i denne matrix. Homeostatiske betingelser styres deposition og nedbrydning af ECM stramt. 5 under bryst tumordannelse, balance af ECM deposition og nedbrydning er forstyrret. Som sådan, er brysttumorer blevet rapporteret at udtrykke rigelige ECM proteiner som collagens, fibronektin og tenascin-C blandt andre. 6 den unormale udtryk for disse proteiner ud over øget mønstre af matrix crosslinking har dokumenteret for at fremme bryst tumor progression, metastaser og terapi modstand1,3, 4,7,8,9.
For at vurdere ECM sammensætning og duktalt morfologi, blev isolation af intakt mælkekirtlerne udført. Her, vi brugte har født hunmus mangelfuld til caveolin-1, en integreret membran protein, som har været forbundet med en aggressiv bryst tumor signatur10,11,12, og kontrollere kvinde har født B6 mus. Histologiske forarbejdning og farvning af disse væv tilladt identifikation af flere ECM proteiner sammen med karakterisering af duktalt morfologi.
Samlet set giver isolering af hele, intakt mælkekirtlerne forskere mulighed for at undersøge vævet hele morfologiske eller cellulære forandringer i svar på eksogene og endogene faktorer. Ulemper ved teknikken, der er knyttet til analyser af 2 dimension (2D) væv sektioner i stedet for en 3D-perspektiv, hvilket ville give et mere komplet billede af den komplekse morfologi af duktalt træet. Betragtning af kompleksiteten af celle-celle og celle-ECM vekselvirkninger, der finder sted i mælkekirtlen, er isolering af hele, intakt kirtler fordelagtige for effektivt analysere duktalt morfologi og protein udtryk i forskellige regioner af den murine brystkirtler kirtel.

<table>
	<tbody>
		<tr>
			<td>Light Microscope</td>
			<td>Olympus</td>
			<td>BX43</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Camera</td>
			<td>Olympus</td>
			<td>DP73</td>
			<td></td>
		</tr>
		<tr>
			<td>Image Analysis Software</td>
			<td>Olympus</td>
			<td>cellSens Entry software</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>NIH</td>
			<td>ImageJ</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5x-R Surgical Micro Loupes</td>
			<td>Rose Micro Solutions</td>
			<td></td>
			<td>Magnification at researcher&#39;s preference</td>
		</tr>
		<tr>
			<td>Mayo Scissors</td>
			<td>Medline</td>
			<td>DYND04035</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Rack</td>
			<td>Fisher Scientific</td>
			<td>121</td>
			<td></td>
		</tr>
		<tr>
			<td>Staining Dish</td>
			<td>Fisher Scientific</td>
			<td>112</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplin Jars</td>
			<td>Fisher Scientific</td>
			<td>19-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td>Size appropriate for tissue</td>
		</tr>
		<tr>
			<td>IHC EnVision+ Kit (HRP, Mouse, DAB+)</td>
			<td>Dako</td>
			<td>K400611-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrosirius Red Kit</td>
			<td>Abcam</td>
			<td>AB150681</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y, alcoholic</td>
			<td>Sigma-Aldrich</td>
			<td>HT110132</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>HHS16</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey Serum</td>
			<td>EMD Millipore</td>
			<td>S30</td>
			<td></td>
		</tr>
		<tr>
			<td>10% Neutral Buffered Formalin</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes, Reagent Grade</td>
			<td>Sigma-Aldrich</td>
			<td>214736</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 200 proof</td>
			<td>Sigma-Aldrich</td>
			<td>792780</td>
			<td>suitable for molecular biology</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline, 1x</td>
			<td>Gibco</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Citrate</td>
			<td>Fisher Scientific</td>
			<td>S279-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>202932</td>
			<td></td>
		</tr>
		<tr>
			<td>Permanent Mounting Medium</td>
			<td>Dako</td>
			<td>S1964</td>
			<td></td>
		</tr>
		<tr>
			<td>Eukitt&#39;s Mounting Medium</td>
			<td>Sigma-Aldrich</td>
			<td>3989</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin antibody</td>
			<td>Abcam</td>
			<td>AB23750</td>
			<td></td>
		</tr>
		<tr>
			<td>Tenascin-C antibody</td>
			<td>Abcam</td>
			<td>AB108930</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha Smooth Muscle Actin antibody</td>
			<td>Abcam</td>
			<td>AB124964</td>
			<td></td>
		</tr>
		<tr>
			<td>Dako Envision Dual Link System HRP</td>
			<td>Dako</td>
			<td>K4065</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of intact, whole mouse mammary glands for analysis of extracellular matrix expression and gland morphology

Målet med denne procedure var at høste #4 abdominal mælkekirtler fra har født hunmus for at vurdere ECM udtryk og duktalt arkitektur. Her, blev en lille lomme under huden oprettet ved hjælp af Mayo saks, giver mulighed for adskillelse af kirtler i det subkutane væv fra den underliggende bughindebetændelse. Visualisering af kirtler blev hjulpet på vej af brugen af 3,5 x-R kirurgisk mikro loupes. Pelt blev omvendt og besejrede tilbage giver identifikation af de intakte brystkirtler fedtpuder. Hver af de #4 abdominal kirtler var ligefremt dissekeret af glidende skalpel klinge lateralt mellem det subkutane lag og kirtler. Straks efter høst, kirtler var placeret i 10% neutrale bufferet formalin til efterfølgende væv forarbejdning. Excision af hele kirtlen er fordelagtig, fordi det primært eliminerer risikoen for at udelukke vigtige væv-wide interaktioner mellem duktalt epitelceller og andre microenvironmental cellular populationer, der kan blive savnet i en delvis biopsi. En ulempe ved metoden er brugen af seriel afsnit fra faste væv, som begrænser analyser af duktalt morfogenese og protein udtryk til diskrete placeringer i kirtlen. Ændringer i duktalt arkitektur og protein udtryk i 3 dimensioner (3D) er som sådan ikke let opnåelige. Samlet, teknikken er gældende for undersøgelser som kræver helt intakt murine mælkekirtlerne for downstream undersøgelser såsom udviklingsmæssige duktalt morfogenese eller bryst kræft.

Hunmus har 5 par af mælkekirtler. Specifikt, er der et par af cervikal kirtler (#1), to par af thorax kirtler (#2 og #3), et par af abdominal kirtler (#4), og 1 par af lyskebrok kirtler (#5) (fig. 1A). Her, isoleret vi #4 kirtler, som de er let identificerbare. I nogle tilfælde blev både #4 og #5 kirtler isoleret sammen som skelnen mellem to var svært. For at isolere intakt #4 abdominal mælkekirtler, pelt var fastgjort og nøje adskilt f...

Watch this Scientific Journal Video about Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology at JoVE.com

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

Vi præsenterer her, en protokol til isolering af hele, intakt mus mælkekirtlerne for at undersøge ekstracellulære matrix (ECM) udtryk og duktalt morfologi. Musen #4 abdominal kirtler blev udvundet fra 8-10 uge gammel kvinde har født mus, fast i neutral bufferet formalin, snit og farves ved hjælp af Immunhistokemi for ECM proteiner.

Isolering af intakt, hele musen mælkekirtlerne for analyse af ekstracellulære Matrix udtryk og morfologi, kirtel

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 ...

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Intraductal Injection for Localized Drug Delivery to the Mouse Mammary Gland

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

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Reconstruction of 3-Dimensional Histology Volume and its Application to Study Mouse Mammary Glands

Genopbygning af 3-dimensionel Histologi Volumen og dens anvendelse til at studere mus mælkekirtler

Histology volume reconstruction facilitates the study of 3D shape and volume change of an organ at the level of macrostructures made up of cells. It can ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

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The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

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