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

Gli autori sono grati per l&#39;impianto nucleo di imaging INRA MIMA2 (INRA, UMR1198, Jouy-en-Josas) e al personale dell&#39;unità IERP (UE 0907, INRA, Jouy-en-Josas) per la cura degli animali e le strutture. Vorremmo anche ringraziare IH Mather, MC Neville e S. Tooze per averci fornito antibodie molto utile.

Topi CD1 sono stati allevati presso l&#39;INRA (UE0907 IERP, Jouy-en-Josas, Francia). Tutti gli aspetti etici della cura degli animali conformi alle relative linee guida ei requisiti di licenza stabiliti dal Ministero dell&#39;Agricoltura francese. Le procedure utilizzate sono state approvate dal comitato locale di etica (accordo 12/097 dal Comethea Jouy-en-Josas / AgroParisTech). 1. ghiandola mammaria preparazione del campione <ol><li> Mouse ghiandola mammaria dissezione <ol><li>...

IHC è un metodo relativamente semplice e diretto sperimentale di localizzare l&#39;antigene in sezioni di tessuto, che dipende principalmente sulle specifiche interazioni epitopi-anticorpo. Anche se un gran numero di protocolli sono utilizzati per localizzare una proteina IIF, il nucleo di queste procedure è quasi sempre la stessa. Tuttavia, vi sono alcuni aspetti critici che possono influenzare fortemente il risultato e deve quindi essere ottimizzati per ciascuno studio individuale IHC. L&#39;aspetto più impegnativo...

La ghiandola mammaria è un atipico organo esocrina mammiferi la cui principale funzione è quella di produrre latte per nutrire i neonati. Lo sviluppo del tessuto mammario si verifica soprattutto dopo la nascita ed è caratterizzata da un processo unico in cui l&#39;epitelio invade lo stroma circostante. Questo tessuto subisce molti cambiamenti (crescita, differenziazione e regressione), soprattutto durante la vita adulta, in concomitanza con variazioni di stato riproduttivo (Figura 1). Oltre alla morfologia generale del tessuto, le proporzioni dei vari tipi di cellule, nonché la loro disposizione all&#39;interno della ghiandola mammaria cambiare radicalmente durante lo sviluppo 1-5. Durante la vita embrionale, l&#39;epitelio mammario deriva da linee latte mammarie, che è definita da un lieve ispessimento e stratificazione della ectoderma, tra gli arti anteriori e posteriori su ciascun lato della linea mediana intorno al giorno embrionale 10.5 (E10.5) (Figura 1A ).Sulla E11.5, la linea del latte scompone in singoli placodi, simmetricamente posizionati lungo la linea di latte mammaria in posizioni riproducibili, e il mesenchima circostante inizia a condensare. I placodi cominciano ad affondare più in profondità nel derma e mesenchima mammaria organizza a strati concentrici intorno al bocciolo mammaria (E12.5-E14.5). A partire dal E15.5, dell&#39;epitelio mammario, comincia a proliferare e allungare a formare il germoglio principale che spinge attraverso il mesenchima mammaria verso il cuscinetto adiposo. Il germoglio primario sviluppa un lume cavo con un&#39;apertura per la pelle, contrassegnata dalla formazione della guaina capezzolo. Sul E18.5, il condotto di allungamento è diventato il cuscinetto adiposo e si è ramificata in un piccolo sistema duttale arborized inglobato nel cuscinetto adiposo. Lo sviluppo è essenzialmente arrestato e la ghiandola mammaria rudimentale rimane morphogenetically quiescente fino alla pubertà. Nel embrione maschio, l&#39;attivazione dei recettori degli androgeni porta alla degenerazione dei germogli, che scompaionoda E15.5. Come di E18, sviluppo mammario cessa fino alla pubertà 6-9. Alla nascita, la ghiandola mammaria ospita un sistema duttale rudimentale che allunga e rami lentamente (una crescita isometrica). All&#39;inizio della pubertà, strutture sferiche situate alle estremità dei condotti chiamati gemme estremità terminale (TEBS), sono formate da uno strato esterno di cellule del cappuccio e un nucleo interno multistrato di cellule (cellule del corpo). Queste strutture sono altamente proliferative e infiltrano il tessuto stromale circostante in risposta a stimoli ormonali. La proliferazione all&#39;interno dei risultati TEBS in allungamento duttale, accoppiato con ramificazione morfogenesi. Questo processo porta alla creazione di una rete arborized epiteliale di base che emana dal capezzolo (Figura 1B, pubertà). A ~ 10-12 settimane dopo la nascita, quando l&#39;epitelio ha invaso tutto il cuscinetto di grasso, la sua espansione si arresta e il TEBS scomparire. Sviluppo duttale subisce quindi cambiamenti dinamici, vale a dire, successive la proliferazione e la regressione delle cellule epiteliali secondo cicli estrali 10 (Figura 1B, adulti). Dall&#39;inizio di gestazione, il tessuto mammario subisce importante crescita e cambiamenti morfologici per preparare l&#39;allattamento. L&#39;epitelio mammario ampiamente proliferano e differenziare, portando ad una ramificata rete altamente tubulo-alveolari. Allo stesso tempo, le cellule epiteliali mammarie (MECS) diventano polarizzata e in grado di sintetizzare e secernere prodotti lattiero-caseari. MECs organizzano in numerose strutture alveolari (acini), che sono circondati da cellule mioepiteliali contrattili e incorporati in uno stroma composta da tessuto connettivo e adiposo, vasi sanguigni e terminazioni nervose (Figura 1B, gravidanza). Inoltre, il lato basale MECs è in stretto contatto con la membrana basale (matrice extracellulare) e le interazioni tra queste due entità strettamente regolano sia la funzione morfogenesi e secretoria della mammellary epitelio 11-13. Tutti questi processi si basano sull&#39;azione di vari segnali ambientali, di cui i più importanti sono hormones14, fattori paracrini e la matrice extracellulare. Ad esempio, progesterone induce vasta ramificazione laterale 15 e alveologenesis che, in combinazione con prolattina (PRL) 16,17, promuove e mantiene la differenziazione degli alveoli. Oltre a steroidi e PRL18, citochine e vie di segnalazione associate allo sviluppo (Wnt e Notch vie di segnalazione) sono anche coinvolti nell&#39;impegno lignaggio mammaria e di sviluppo 19-21. Al termine della gravidanza, i MECs luminali iniziano a produrre un latte ricco di proteina nota come colostro nel lume degli alveoli. Inoltre, il progesterone agisce sulla permeabilità epiteliale e dato che le giunzioni strette sono ancora aperte, il colostro si trova anche nel sangue materno. Dopo il parto, il Mammary epitelio occupa quasi tutto il volume della ghiandola mammaria e è altamente organizzato (Figura 2, mammario). Unità latte produzione, ovvero alveoli (Figura 2, alveoli), sono formate da un monostrato di polarizzati mammarie epiteliali cellule secretorie (MESCs), con la loro membrana plasmatica apicale delimita il lume. Alveoli si dispongono in lobuli che sono raggruppati in lobi collegati a condotti che drenano il latte con l&#39;ambiente esterno (Figura 2, del lobo). Allattamento si verifica, ad esempio., MESCs iniziano a secernere abbondanti quantità di latte, innescato principalmente dal calo di ormoni della placenta (principalmente progesterone) (Figura 1B, allattamento). Geni delle proteine ​​del latte sono attivati ​​in un corso a tempo temporale definito che va dalla gravidanza al allattamento 9,22,23, soprattutto in risposta a ipofisaria PRL rilasciato al momento dell&#39;allattamento. Allo stesso tempo, i contatti tra MESCs e la matrice extracellulare, sia stimolano proteine ​​del latte syntESI attraverso i segnali che sono mediati attraverso le interazioni tra integrine e laminina 24,25 cellulari, e sopprimono l&#39;apoptosi in MESCs 26,27. Queste vie di segnalazione provocano l&#39;attivazione di proteine ​​del latte gene promoters 28 attraverso l&#39;attivazione della trascrizione specifici fattori di 29. Contatti cellula-cellula sono importanti anche per alcuni aspetti della differenziazione tra cui l&#39;istituzione della polarità apicale e la secrezione vettoriale di prodotti lattiero-caseari. Giunzioni strette rapidamente stretti dopo l&#39;inizio della lattazione e MESCs finemente orchestrano l&#39;assorbimento di molecole dal sangue così come la sintesi, il trasporto e la secrezione di componenti del latte, in risposta alle esigenze nutrizionali dei neonati. Al momento della suzione, la contrazione delle cellule mioepiteliali circondano gli alveoli si verifica in risposta a ossitocina e porta alla emissione del latte attraverso i condotti e nel capezzolo. Il latte è un liquido che contiene proteine ​​complessa (principalmentecaseine), zuccheri (principalmente lattosio), lipidi e minerali, come pure molecole bioattive come immunoglobuline A (IgA), fattori di crescita e ormoni. Sono sintetizzati caseine, assemblati in strutture supramolecolari, ossia micelle di caseina, trasportate lungo la via secretoria, e poi rilasciato per esocitosi, cioè, la fusione delle vescicole secretorie-caseina contenente (SV) con la membrana plasmatica apicale di MESC (Figura 2). Traffico intracellulare si basa su scambi materiali tra compartimenti membranose e coinvolge solubile N-ethylmaleimide sensibile Fusion (NSF) Allegato Protein (SNAP) Receptor (SNARE) 30,31. La famiglia delle proteine ​​SNARE è suddivisa in SNAREs vescicolari (v-lacci), presenti nella membrana della vescicola e SNAREs bersaglio (t-lacci), localizzati sulle membrane di destinazione. Con sfreccia tra loro domini coiled-coil, V e t-SNARE assemblare per formare un complesso altamente stabile fascio quattro elica, indicati come thcomplesso SNARE e. Questo complesso promuove la fusione di due doppi strati lipidici opposti gradualmente portarli in prossimità 30,32. In seguito, i complessi SNARE sono dissociate dalla adenosina trifosfatasi NSF e le sue proteine ​​adattatore Snap e SNARE proteine ​​sono riciclati di nuovo al loro compartimento di origine 33. È interessante notare che, ogni proteina SNARE risiede prevalentemente in compartimenti cellulari distinti e rullante accoppiamento possono contribuire alla specificità degli eventi di fusione intracellulari 34. Precedenti studi suggeriscono che almeno Protein 23 (SNAP23) e vescicole associata a membrana di proteine ​​8 (VAMP8), e sintaxine (Stx) sinaptosomiali associata -7 e -12 svolgono un ruolo nella caseina esocitosi 35,36. Queste proteine ​​sono state trovate anche in associazione con la frazione lipidica del latte, vale a dire, di grasso del latte (globuli MFGs) 37. Il modello prevalente corrente postula che gocce lipidiche citoplasmatici (CLD) sono formati dall&#39;accumulo di l neutroipids (soprattutto trigliceridi ed esteri di steroli) e colesterolo deriva dalla dieta materna tra i due volantini del reticolo endoplasmatico (ER) membrane 38-41. Grandi CLD si formano, almeno in parte, dalla fusione di CLD piccole mentre viene trasportato al lato apicale MESCs dove vengono rilasciati come MFGs (1-10 micron di diametro) per gemmazione, essendo avvolto dalla membrana plasmatica apicale MESC 40-42. Allattamento cessa dopo cuccioli sono svezzati e le MESCs progressivamente muoiono per apoptosi, che porta alla regressione del tessuto mammario a uno stato puberale (Figura 1B, involuzione). Immunofluorescenza (IF) è un metodo di laboratorio di analisi comune usato in quasi tutti gli aspetti della biologia, sia nella ricerca e nella diagnostica clinica. SE tecniche possono essere eseguite su sezioni di tessuto (immunoistochimica, IHC) o cellulari (immunocitochimica, ICC) campioni. Questo approccio potente si basa sull&#39;utilizzo di fluorescente-anticorpi marcati che specificamente si legano (direttamente o indirettamente) all&#39;antigene di interesse, permettendo così la visualizzazione della sua distribuzione tissutale tramite microscopia a fluorescenza. Segnali di fluorescenza per lo più dipendono dalla qualità e la concentrazione degli anticorpi e corretta manipolazione del campione. Un protocollo semplice immunofluorescenza indiretta (IIF) viene presentato per rilevare prodotti lattiero-caseari (caseine e MFGs) e proteine ​​coinvolte nella secrezione di prodotti lattiero-caseari (butirofilina (BTN1), rullante proteine) su sezioni congelate di tessuto mammario del mouse (Figura 3). Anche se questo protocollo offre una panoramica completa IHC, che vanno dalla raccolta di tessuto per l&#39;immagine dopo il trattamento, critica e passaggi facoltativi, nonché alcune raccomandazioni tecniche vengono anche presentati e discussi.

<table border="0" cellpadding="0" cellspacing="0" style="width:756px;" width="755">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Dissection</td>
			<td style="width:155px;">Company</td>
			<td style="width:127px;">Catalog Number</td>
			<td style="width:236px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Pins</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ethanol</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Scissors</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel and adapted blades</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Ice</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Towel paper</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Tissue sample preparation</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;">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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OCT compound/Tissue Tek</td>
			<td style="width:155px;">Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Liquid nitrogen</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<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>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Slide box</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Brushes</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Permanent marker (black)</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:239px;">Nail polish</td>
			<td style="width:155px;"></td>
			<td></td>
			<td></td>
		</tr>
		<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>
			<td style="width:127px;">Catalog Number</td>
			<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

L&#39;immunofluorescenza indiretta è utilizzato per rilevare e localizzare le proteine ​​di interesse in un tessuto. Il protocollo qui presentato descrive un metodo semplice e completo per la rilevazione di proteine ​​immunitario, mouse allattamento ghiandola mammaria essere preso come esempio. Un protocollo per la preparazione dei campioni di tessuto, soprattutto per quanto riguarda la dissezione del mouse di ghiandola mammaria, la fissazione dei tessuti e sezionamento tessuti congelati, sono dettagliati. Un protocollo standard per eseguire immunofluorescenza indiretta, tra cui un passo di recupero dell&#39;antigene opzionale, è anche presentato. L&#39;osservazione delle sezioni di tessuto etichettati così come l&#39;acquisizione di immagini e post-trattamenti sono anche indicati. Questa procedura fornisce una panoramica completa, dalla raccolta di tessuto animale per la localizzazione cellulare di una proteina. Anche se questo metodo generale può essere applicato ad altri campioni di tessuto, deve essere adattato ad ogni coppia anticorpo tessuto / primary studiato.

La ghiandola mammaria è una ghiandola sottocutanea situato lungo la struttura ventrale sia del torace e dell&#39;addome nei roditori. La posizione dei cinque coppie di ghiandole del mouse durante la gestazione è mostrata in Figura 4. La morfologia della ghiandola mammaria cambia drasticamente durante il suo sviluppo, riflettendo modifiche funzionali richieste per prepararsi alla piena lattazione (Figura 1B). Negli animali vergini o nullipare, la ghiandola...

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

Il protocollo immunofluorescenza indiretta descritto in questo articolo consente il rilevamento e la localizzazione delle proteine ​​nella ghiandola mammaria del mouse. Un metodo completo è dato per la preparazione di campioni della ghiandola mammaria, per eseguire immunoistochimica, per l&#39;immagine delle sezioni di tessuto al microscopio a fluorescenza, e di ricostruire le immagini.

Immunofluorescenza indiretta su sezioni congelate di mouse ghiandola mammaria

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.

Immunoistochimica su sezioni di paraffina di epidermide mouse utilizzando anticorpi fluorescenti

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

Ricerca

Biologia

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.

Topo cellule epiteliali mammarie forma Mammospheres durante la differenziazione lattogenica

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.

Cambiamenti nella morfologia della ghiandola mammaria e rischio di cancro al seno in topi

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.

Sezioni incluse in paraffina e surgelati di Drosophila Adulti Muscoli

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.

Isolamento di cellule staminali del mouse ghiandole salivari

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.

Valutazione dello Sviluppo ghiandola mammaria e funzione in modelli murini

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.

Cella singola Profiling trascrizionale dei cardiomiociti adulti mouse

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.

Iniezione intraduttale per Localizzato Drug Delivery per il mouse ghiandola mammaria

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.

Effetto di Male Accessory Gland prodotti in deposizione delle uova in gasteropodi

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.

Isolamento delle ghiandole mammarie Mouse intatto, intero per l'analisi di espressione di matrice extracellulare e la morfologia della ghiandola

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 ...