Name: indirect immunofluorescence on frozen sections of mouse mammary gland
Uploaded: 2015-12-01T14:00:00
Description: Watch this Scientific Journal Video about Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland at JoVE.com

De auteurs zijn dankbaar voor de INRA MIMA2 imaging kernfaciliteit (INRA, UMR1198, Jouy-en-Josas) en aan het personeel van de IERP eenheid (UE 0907, INRA, Jouy-en-Josas) voor dierlijke zorg en voorzieningen. We willen ook graag IH Mather, MC Neville en S. Tooze bedanken voor ons zeer nuttig antibodie.

CD1 muizen werden gefokt bij INRA (UE0907 IERP, Jouy-en-Josas, Frankrijk). Alle ethische aspecten van de verzorging van de dieren voldoet aan de relevante richtlijnen en eisen vergunningverlening door het Franse ministerie van Landbouw vastgelegd. De procedures die zijn goedgekeurd door de lokale ethische commissie (overeenkomst 12/097 van de Comethea Jouy-en-Josas / AgroParisTech). 1. borstklier Monstervoorbereiding <ol><li> Muis borstklier dissectie <ol><li> Euthanaseren muizen ...

IHC is een relatief eenvoudige en eenvoudige experimentele methode om antigeen in weefselcoupes, die vooral afhankelijk specifiek epitoop-antilichaam interacties te lokaliseren. Hoewel een groot aantal protocollen worden gebruikt om een ​​proteïne te lokaliseren door IIF, de kern van deze procedures is vrijwel altijd gelijk. Er zijn echter enkele kritische aspecten die sterk kunnen beïnvloeden het resultaat en moet dus worden geoptimaliseerd voor elke afzonderlijke IHC studie. De meest uitdagende aspect van deze b...

De borstklier is een atypische zoogdieren exocrine orgaan waarvan de belangrijkste functie is om melk te produceren pasgeborenen te voeden. De ontwikkeling van de borstklier weefsel komt vooral na de geboorte en wordt gekenmerkt door een unieke werkwijze, waarbij het epitheel binnendringt het omliggende stroma. Dit weefsel ondergaat vele veranderingen (groei, differentiatie en regressie), met name tijdens het volwassen leven, gelijktijdig met variaties in voortplantingsstatus (figuur 1). Naast de algemene morfologie van het weefsel was het aantal verschillende celtypen en hun rangschikking in de melkklier drastisch veranderen tijdens de ontwikkeling 1-5. Tijdens de embryonale leven, de mammaire epitheel afgeleid van mammaire melkleidingen, gedefinieerd door een lichte verdikking en stratificatie van het ectoderm, tussen de voor- en achterpoten aan weerszijden van de middellijn ongeveer embryonale dag 10,5 (E10,5) (Figuur 1A ).Op E11.5, de melk lijn breekt in individuele placodes, die symmetrisch zijn geplaatst langs de borstklier melk lijn op reproduceerbare locaties en de omringende mesenchym begint te condenseren. De placodes beginnen om dieper in de lederhuid en de borstklier mesenchym organiseert in concentrische lagen rond de borstklieren kiem (E12.5-E14.5). Vanaf E15.5, de borstklier epitheel, begint te groeien en te verlengen om de primaire spruit die duwt door de borstklier mesenchym naar het vet pad vormen. De primaire spruit ontwikkelt een hol lumen met een opening aan de huid, gekenmerkt door de vorming van de tepel huls. Op E18.5, heeft het verlengen van duct uitgegroeid tot het vet pad en heeft vertakt in een kleine arborized ductaal systeem omvatte in het vet pad. Ontwikkeling is in wezen gearresteerd en de rudimentaire borstklier blijft morfogenetisch rustig tot de puberteit. In het mannelijke embryo, het activeren van androgene receptoren leidt tot de degeneratie van de knoppen die verdwijnendoor E15.5. Vanaf E18, borstklier ontwikkeling ophoudt tot de puberteit 6-9. Bij de geboorte, de melkklier herbergt een rudimentaire ductaal systeem dat verlengt en takken langzaam (isometrische groei). Aan het begin van de puberteit, bolvormige structuren op de uiteinden van de kanalen genoemd uiteinde knoppen (Tebs), gevormd uit een buitenlaag van cellen cap en een meerlagig kern van cellen (lichaamscellen). Deze structuren zijn zeer proliferatieve en infiltreren de omringende stromale weefsel in reactie op hormonale signalen. Proliferatie binnen de TEBS resultaten in ductale rek combinatie met vertakkende morfogenese. Dit proces leidt tot de oprichting van een basis epitheliale arborized netwerk afkomstig van de tepel (Figuur 1B, de puberteit). Bij ~ 10-12 weken na de geboorte, wanneer het epitheel van de hele dikke pad is binnengedrongen, stopt en de uitbreiding van de TEBS verdwijnen. Ductaal ontwikkeling ondergaat dynamische veranderingen, dat wil zeggen, Successive proliferatie en regressie van epitheliale cellen volgens oestrische cycli 10 (Figuur 1B, volwassene). Vanaf het begin van de zwangerschap, het borstweefsel ondergaat belangrijke groei en morfologische veranderingen te bereiden lactatie. De borstklier epitheel uitgebreid prolifereren en differentiëren, leidt tot een sterk vertakte tubulo-alveolaire netwerk. Gelijktijdig, borstklieren epitheelcellen (MEC) worden gepolariseerd en in staat om synthetiseren en uitscheiden melkproducten. MEC organiseren in verschillende celstructuren (acini) die zijn omgeven door contractiele myo cellen en opgenomen in een stroma bestaande uit bind- en vetweefsel, bloedvaten en zenuwuiteinden (Figuur 1B, zwangerschap). Bovendien is de basale zijde van MEC in nauw contact met de basaalmembraan (extracellulaire matrix) en interacties tussen deze twee entiteiten vast te reguleren zowel morfogenese en secretoire functie van het mammary epitheel 11-13. Al deze werkwijzen vertrouwen op de werking van verschillende omgevingsfactoren, waarvan de belangrijkste zijn hormones14, paracriene factoren en de extracellulaire matrix. Bijvoorbeeld, progesteron veroorzaakt uitgebreide-side vertakking 15 en alveologenesis dat, in combinatie met prolactine (PRL) 16,17 bevordert en handhaaft de differentiatie van de alveoli. Naast steroïden en PRL18, cytokines en signaalwegen geassocieerd met de ontwikkeling (Wnt en Notch signaalroutes) zijn betrokken bij mammaire lineage betrokkenheid en ontwikkeling 19-21. Aan het einde van de zwangerschap, het luminale MEC&#39;s ​​beginnen een eiwitrijke melk bekend als biest in het lumen van de alveoli produceren. Bovendien progesteron werkt op de epitheliale permeabiliteit en aangezien de tight junctions nog open is colostrum ook in het maternale bloed. Na de bevalling, de Mammary epitheel neemt bijna alle borstklier volume en sterk georganiseerd (Figuur 2, borstklier epitheel). Melkproducerende eenheden, namelijk alveoli (figuur 2, alveole), gevormd door een monolaag van gepolariseerde mammaire uitscheidende epitheelcellen (mESCs), met hun apicale plasmamembraan afbakening van het lumen. Alveoli rangschikken zichzelf in lobben die zijn gegroepeerd in lobben verbonden met leidingen die melk weglopen naar buiten milieu (figuur 2, kwab). Borstvoeding optreedt, dwz., MESCs beginnen om overvloedige hoeveelheden melk, voornamelijk veroorzaakt door de daling van de placenta hormonen (voornamelijk progesteron) (Figuur 1B, borstvoeding) afscheiden. Melkeiwitten genen geactiveerd in een gedefinieerde tijd tijdsverloop variërend van zwangerschap tot borstvoeding 9,22,23, vooral in reactie op hypofyse PRL vrijgegeven op het moment van zogen. Gelijktijdig, contacten tussen mESCs en de extracellulaire matrix zowel stimuleren melkeiwit synthesis door de signalen die worden bemiddeld via de interacties tussen cellulaire integrines en laminine 24,25 en onderdrukken apoptose in mESCs 26,27. Deze signaleringsroutes leidt tot activering van melkeiwit genpromoters 28 door de activering van specifieke transcriptiefactoren 29. Cel-cel contacten zijn ook belangrijk voor een aantal aspecten van differentiatie waaronder de oprichting van apicale polariteit en de vectoriële afscheiding van melkproducten. Tight junctions snel sluiten na het begin van de lactatie en mESCs fijn orkestreren de opname van moleculen uit het bloed en de synthese, transport en uitscheiding van melkbestanddelen, in reactie op de voedingsbehoeften van pasgeborenen. Ten tijde van het zuigen, de samentrekking van de myo-epitheliale cellen rondom de alveoli optreedt in reactie op oxytocine en leidt tot melksecretie door de kanalen en naar de tepel. Melk is een complexe vloeistof die eiwitten bevat (meestalcaseïnen), suikers (hoofdzakelijk lactose), lipiden en mineralen, alsmede biologisch actieve moleculen zoals immunoglobulinen A (IgA), groeifactoren en hormonen. Caseïne worden gesynthetiseerd, geassembleerd in supramoleculaire structuren, namelijk caseïne micellen, langs de secretieroute getransporteerd, en vervolgens vrijgegeven door exocytose, dwz de fusie van caseïne bevattende secretorische blaasjes (SV&#39;s) met de apicale plasmamembraan van MESC (figuur 2). Intracellulaire verkeer is gebaseerd op materiële uitwisseling tussen membraneuze compartimenten en het gaat Oplosbare N-ethylmaleïmide-Sensitive Fusion (NSF) Attachment Protein (SNAP) Receptor (SNARE) 30,31. Het SNARE eiwitten familie onderverdeeld in vesiculaire SNAREs (v-SNAREs), aanwezig in het vesicle membraan, en target SNAREs (t-SNAREs), gelokaliseerd op het doel membranen. Door snellen door hun coiled-coil domeinen, v- en t-SNAREs monteren op een zeer stabiele vier-helix bundel te vormen, aangeduid als the SNARE complex. Dit complex bevordert de fusie van twee tegengestelde lipidendubbellagen door hen geleidelijk aan te brengen in de nabijheid 30,32. Daarna worden SNARE complexen gedissocieerd door het NSF adenosinetrifosfatase en de adapter eiwit SNAP en SNARE eiwitten worden teruggevoerd naar hun compartiment van herkomst 33. Interessant is dat elke SNARE eiwit woont voornamelijk in verschillende cellulaire compartimenten en snare paring kan bijdragen aan de specificiteit van intracellulaire fusie gebeurtenissen 34. Eerdere studies suggereren dat tenminste Synaptosomale-geassocieerd eiwit 23 (SNAP23) en bolletjes-geassocieerde membraaneiwit 8 (VAMP8) en syntaxins (Stx) -7 en -12 spelen een rol in caseïne exocytose 35,36. Deze eiwitten zijn ook gevonden in associatie met de lipidefractie van melk, dat wil zeggen, melkvetbolletjes (MFGs) 37. De huidige heersende model stelt dat cytoplasmatische vetdruppels (CLD&#39;s) worden gevormd door de ophoping van neutrale lipids (voornamelijk triacylglycerolen en sterolesters) en cholesterol verkregen uit de voeding van de moeder tussen twee bladen van het endoplasmatisch reticulum (ER) membraan 38-41. Grote CLDs worden gevormd, ten minste gedeeltelijk, door de fusie van kleinere CLDs terwijl aan de apicale zijde van mESCs waar ze worden vrijgelaten als MFGs (1-10 um in diameter) door knopvorming vervoerd, wordt omhuld door de MESC apicale plasmamembraan 40-42. Borstvoeding ophoudt nadat pups gespeend en mESCs geleidelijk sterven apoptose leiden tot de regressie van de zoogdierweefsel naar een puberale toestand (figuur 1B, involutie). Immunofluorescentie (IF) is een gemeenschappelijk analytisch laboratorium methode die wordt gebruikt in bijna alle aspecten van de biologie, zowel in onderzoek en klinische diagnostiek. IF technieken kunnen worden uitgevoerd op weefselcoupes (immunohistochemie, IHC) of cellen (immunocytochemie, ICC) monsters. Deze krachtige aanpak berust op het gebruik van fluorescent-gemerkte antilichamen die specifiek binden (direct of indirect) aan het antigeen van belang, waardoor de visualisatie van de weefselverdeling met fluorescentiemicroscopie. Fluorescentiesignalen vooral afhankelijk van de kwaliteit en concentratie van de antilichamen en juiste hantering van het monster. Een eenvoudige indirecte immunofluorescentie (IIF) protocol wordt voorgelegd aan zuivelproducten (caseïne en MFGs) en eiwitten die betrokken zijn bij melkproduct secretie detecteren (butyrofiline (btn1), snare eiwitten) op bevroren delen van muizen borstweefsel (figuur 3). Hoewel dit protocol een volledig overzicht IHC, van weefselverzameltoestel aan te nabehandeling, kritisch en optionele stappen en enkele technische aanbevelingen worden ook besproken.

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			<td height="21" style="height:21px;width:239px;">Dissection</td>
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			<td height="21" style="height:21px;width:239px;">Pins</td>
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			<td height="21" style="height:21px;width:239px;">Ethanol</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Scissors</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Scalpel and adapted blades</td>
			<td style="width:155px;"></td>
			<td></td>
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			<td height="21" style="height:21px;width:239px;">Ice</td>
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			<td height="21" style="height:21px;width:239px;">Towel paper</td>
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			<td height="21" style="height:21px;width:239px;">Tissue sample preparation</td>
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			<td>Comments/Description</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Phosphate Buffered Saline (pH7.4)</td>
			<td style="width:155px;">Sigma</td>
			<td>P-3813</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Paraformaldehyde (PFA, 32% EM grade, 100 ml)</td>
			<td style="width:155px;">Electron Microscopy Sciences</td>
			<td>15714-S</td>
			<td>personnal protection equipment required WARNING: this product will expose you to Formaldehyde Gas, a chemical known to cause cancer</td>
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		<tr height="21">
			<td height="21" style="height:21px;">OCT compound/Tissue Tek</td>
			<td style="width:155px;">Sakura</td>
			<td>4583</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Sucrose (D-saccharose)</td>
			<td style="width:155px;">VWR</td>
			<td>27480.294</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Plastic molds</td>
			<td style="width:155px;">Dominique Dutscher</td>
			<td>39910</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Liquid nitrogen</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Cryostat/sample support</td>
			<td style="width:155px;">Leica&#160;</td>
			<td>CM3050S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Razor blades (SEC35)</td>
			<td style="width:155px;">Thermo Scientific</td>
			<td>152200</td>
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			<td height="21" style="height:21px;width:239px;">Slide box</td>
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			<td height="42" style="height:42px;width:239px;">Glass slides Superfrost/Superfrost Ultra Plus</td>
			<td style="width:155px;">Thermo Scientific</td>
			<td>10143560W90/1014356190</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Brushes</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">IHC</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Super Pap Pen</td>
			<td style="width:155px;">Sigma</td>
			<td>Z377821-1EA</td>
			<td></td>
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			<td height="21" style="height:21px;width:239px;">Permanent marker (black)</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
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		<tr height="25">
			<td height="25" style="height:25px;width:239px;">50 mM NH4Cl in PBS</td>
			<td style="width:155px;">Sigma</td>
			<td>A-0171</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">0.1 M glycine in PBS</td>
			<td style="width:155px;">VWR</td>
			<td>24403.367</td>
			<td></td>
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		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Antigen Retrieval solution: Tris 100 mM 5% urea pH9.6</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Heater (up to 100&#176;C)</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:155px;">Sigma</td>
			<td>A7906-100G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Vectashield (anti-fading mounting medium) without DAPI/with DAPI</td>
			<td style="width:155px;">Vector Laboratories</td>
			<td>H-1000/H-1200</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Glass coverslips 22x50mm (microscopy grade)</td>
			<td style="width:155px;">VWR</td>
			<td>CORN2980-225</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nail polish</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Primary antibodies</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:239px;">Rabbit anti-mouse caseins (#7781; 1:50 dilution)</td>
			<td style="width:155px;">generously gifted by M.C. Neville (University of Colorado Health Sciences 
			Center, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Mouse anti-cytokeratin 8 (CK8, clone 1E8, 1:50 dilution)</td>
			<td style="width:155px;">Biolegend (Covance)</td>
			<td>MMS-162P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Mouse anti-cytokeratin 14 (CK14, cloneLL002, 1:50 dilution)</td>
			<td style="width:155px;">Thermo Scientific</td>
			<td>MS-115-P0/P1</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:239px;">Rabbit anti-butyrophilin (1:300 dilution)</td>
			<td style="width:155px;">generously gifted by I.H. Mather (Department of Animal and Avian Sciences University of Maryland College Park, USA)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:239px;">Rabbit anti-Stx6 (1:50 dilution)</td>
			<td style="width:155px;">generously gifted S. Tooze 
			(Cancer Research UK, London Research Institute, London, UK)</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Rabbit anti-VAMP4 (1:50 dilution)</td>
			<td style="width:155px;">Abcam</td>
			<td>ab3348</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Secondary antibodies</td>
			<td style="width:155px;">Company</td>
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			<td>Comments/Description</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;">Rhodamine-conjugated goat anti-rabbit IgG (H + L) (1:300 dilution)</td>
			<td style="width:155px;">Jackson ImmunoResearch Laboratories</td>
			<td>111-025-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Counterstains</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">Bodipy 493/503</td>
			<td style="width:155px;">Life Technologies (Molecular Probes)</td>
			<td>D-3922</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">DAPI (4-6-diamidino-2-phenylindole)</td>
			<td style="width:155px;">Life Technologies (Molecular Probes)</td>
			<td>D-1306</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Observation/Image capture</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:239px;">conventional fluorescence microscope</td>
			<td style="width:155px;">Leica Leitz DMRB 
			microscope</td>
			<td></td>
			<td style="width:236px;">Standard filters for FITC, Rhodamine 
			and DAPI emissions,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; &#215;63 oil-immersion objective (NA 1.3), DP50 imaging camera (Olympus), Cell&#710;F software (Olympus)</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:239px;">Laser Scanning Microscope (confocal microscopy)</td>
			<td style="width:155px;">Zeiss LSM 
			510 microscope</td>
			<td></td>
			<td style="width:236px;">Plan-Apochromat &#215;63 oil-immersion objective (NA 1.4),&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; CLSM 510 software,&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Confocal facilities, MIMA2 Platform, INRA Jouy-en-Josas, France, http://mima2.jouy.inra.fr/mima2)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Image treatment</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ 1.49k software</td>
			<td style="width:155px;"></td>
			<td></td>
			<td>Free software</td>
		</tr>
	</tbody>
</table>

indirect immunofluorescence on frozen sections of mouse mammary gland

Indirecte immunofluorescentie wordt gebruikt voor het opsporen en lokaliseren eiwitten van belang in een weefsel. De hier gepresenteerde protocol beschrijft een volledige en eenvoudige methode voor het immuunsysteem detectie van eiwitten, de muis lacterende borstklier genomen als voorbeeld. Een protocol voor de voorbereiding van de weefselmonsters, met name met betrekking tot de ontrafeling van de muis borstklier, weefsel fixatie en ingevroren weefsel snijden, zijn gedetailleerd. Een standaard protocol voor indirecte immunofluorescentie voeren, inclusief een optionele antigen retrieval stap wordt gepresenteerd. De waarneming van de gelabelde coupes, alsmede het verwerven en post-behandelingen worden ook vermeld. Deze procedure geeft een volledig overzicht van het verzamelen van dierlijk weefsel om de cellulaire lokalisatie van een eiwit. Hoewel dit algemene werkwijze kan worden toegepast op andere weefselmonsters, moet worden aangepast aan elk weefsel / primair antilichaam paar onderzocht.

De borstklier is een klier onderhuids gelegen langs de ventrale constructie van zowel de thorax en de buik bij knaagdieren. De ligging van de vijf paren van klieren van de muis tijdens dracht wordt weergegeven in figuur 4. De morfologie van de melkklier drastisch verandert tijdens de ontwikkeling, hetgeen functionele aanpassingen nodig oog op een volledige lactatie (Figuur 1B). In maagdelijke of nullipaar dieren, de borstklier bestaat uit een dun vertakte d...

Watch this Scientific Journal Video about Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland at JoVE.com

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

De indirecte immunofluorescentie protocol beschreven in dit artikel laat de detectie en de lokalisatie van eiwitten in de muis borstklier. Een complete methode wordt gegeven voor te bereiden borstklier monsters, om immunohistochemie uit te voeren, om de afbeelding van het weefsel secties met behulp van fluorescentie microscopie, en om beelden te reconstrueren.

Indirecte Immunofluorescentie op Bevroren Secties van Muis borstklier

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

indirect-immunofluorescence-on-frozen-sections-of-mouse-mammary-gland

Research

JoVE Journal

Biology

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

Immunohistochemie op paraffine secties van Mouse Opperhuid gebruik van fluorescerende antilichamen

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

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.

Muis uier epitheelcellen vorm Mammospheres Tijdens lactogenic Differentiatie

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.

Veranderingen in de borstklier Morfologie en het risico op borstkanker bij ratten

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 ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

Paraffine ingebedde en vriescoupes van Drosophila Adult Spieren

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

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.

Isolatie van Mouse speekselklier 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 ...

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.

Evaluatie van de ontwikkeling van de borstklier en functie in muismodellen

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 ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Single Cell Transcriptionele Profilering van de volwassen muis cardiomyocyten

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

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.

Intraductale Injectie voor Gelokaliseerde Drug Delivery naar de muis borstklier

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

Effect of Male Accessory Gland Products on Egg Laying in Gastropod Molluscs

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and activating sperm, the components in the seminal fluid can also influence female physiology to augment fertilization success of the sperm donor. While many studies have reported such effects in species with separate sexes, few studies have addressed this in simultaneously hermaphroditic animals. This video protocol presents a method to study effects of seminal fluid in gastropods, using a simultaneously hermaphroditic freshwater snail, the great pond snail Lymnaea stagnalis, as model organism. While the procedure is shown using complete prostate gland extracts, individual components (i.e., proteins, peptides, and other compounds) of the seminal fluid can be tested in the same way. Effects of the receipt of ejaculate components on egg laying can be quantified in terms of frequency of egg laying and more subtle estimates of female reproductive performance such as egg numbers within each egg masses. Results show that seminal fluid proteins affect female reproductive output in this simultaneous hermaphrodite, highlighting their importance for sexual selection.

This video protocol demonstrates a method to study effects of seminal fluid in gastropods, using the hermaphroditic freshwater snail Lymnaea stagnalis.

Effect van Accessory Gland producten op eierleggende in buikpotigen

In internally fertilizing animals, seminal fluid is usually added to the spermatozoa, together forming the semen or ejaculate. Besides nourishing and ...

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.

Isolatie van Intact, hele muis borstklieren p.a. van extracellulaire Matrix expressie en klier morfologie

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 ...