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

Os autores agradecem à facilidade núcleo imagiologia INRA MIMA2 (INRA, UMR1198, Jouy-en-Josas) e ao pessoal da unidade IERP (UE 0907, INRA, Jouy-en-Josas) por cuidar dos animais e instalações. Gostaríamos também de agradecer a IH Mather, MC Neville e S. Tooze por nos fornecer antibodie muito útil.

Ratinhos CD1 foram criados pelo INRA (UE0907 IERP, Jouy-en-Josas, França). Todos os aspectos éticos de cuidados com animais cumprido com as orientações pertinentes e requisitos de licenciamento estabelecidos pelo Ministério da Agricultura francês. Os procedimentos utilizados foram aprovados pelo comitê de ética local (12/097 acordo do Comethea Jouy-en-Josas / AgroParisTech). 1. glândula mamária Preparação de Amostras <ol><li> Glândula mamaria do rato dissecção <ol><...

IHC é um método experimental simples e relativamente simples para localizar antigénio em secções de tecido, o qual depende primariamente em interacções epitopo-anticorpo específicos. Embora um grande número de protocolos são utilizados para localizar uma proteína por IFI, a essência destes procedimentos é quase sempre o mesmo. No entanto, existem alguns aspectos críticos que podem influenciar fortemente o resultado e, por conseguinte, devem ser optimizadas para cada estudo IHC indivíduo. O maior desafio d...

A glândula mamária é um órgão de mamíferos exócrina atípica cuja principal função é produzir leite para alimentar recém-nascidos. O desenvolvimento do tecido mamário ocorre principalmente após o nascimento e é caracterizada por um processo único, em que o epitélio invade o estroma circundante. Este tecido sofre muitas mudanças (crescimento, diferenciação e regressão), especialmente durante a vida adulta, concomitantemente com variações no estado reprodutivo (Figura 1). Além da morfologia geral do tecido, as proporções de tipos de células diferentes, bem como a sua disposição no interior da glândula mamaria mudar dramaticamente durante o desenvolvimento 1-5. Durante a vida embrionária, o epitélio mamário deriva de linhas de leite mamarias, que é definido por um ligeiro espessamento e estratificação da ectoderme, entre as partes dianteiras e traseiras dos membros de cada lado da linha média por volta do dia embrionário 10,5 (E10.5) (Figura 1A ).Em E11.5, a linha do leite se divide em placodes individuais, que são posicionados simetricamente ao longo da linha de leite mamário em locais reprodutíveis, eo mesênquima circundante começa a condensar. Os placodes começar a afundar-se mais profundamente na derme e no mesênquima mamária organiza em camadas concêntricas ao redor do broto mamário (E12.5-E14.5). Em E15.5, o epitélio mamário, começa a proliferar e alongada para formar o rebento principal que empurra através do mesênquima mamária para a almofada de gordura. O rebento principal desenvolve um lúmen oco com uma abertura para a pele, caracterizado por a formação da bainha de mamilo. Em E18.5, o ducto alongando tem crescido na almofada de gordura e tem ramificações em um pequeno sistema ductal arborized englobadas na almofada de gordura. O desenvolvimento é essencialmente preso e da glândula mamária rudimentar permanece morphogenetically de repouso até a puberdade. No embrião do sexo masculino, a activação de receptores de androgénio conduz à degeneração dos gomos, que desaparecempor E15.5. Em E18, desenvolvimento mamário cessa puberdade até 6-9. Ao nascer, a glândula mamária abriga um sistema ductal rudimentar que alonga e ramos lentamente (crescimento isométrico). No início da puberdade, as estruturas esféricas localizadas nas pontas dos ductos chamado as papilas extremidade terminal (TEBS), são formadas por uma camada externa de células do tampão e um núcleo interior de várias camadas de células (células do corpo). Estas estruturas são altamente proliferativo e infiltrar o tecido do estroma circundante, em resposta a estímulos hormonais. Proliferação dentro dos resultados TEBS em alongamento ductal, juntamente com ramificação morfogênese. Este processo leva ao estabelecimento de uma rede de base epitelial arborized que emana do bocal (Figura 1B, a puberdade). A ~ 10-12 semanas após o nascimento, quando o epitélio invadiu toda a almofada de gordura, a sua expansão pára e a TEBS desaparecer. Desenvolvimento ductal, em seguida, passa por mudanças dinâmicas, ou seja, successive proliferação e regressão de células epiteliais de acordo com ciclos estrais 10 (Figura 1B, adulto). Desde o início da gestação, o tecido mamário de crescimento sofre alterações morfológicas e importante para se preparar para a lactação. O epitélio mamário extensivamente proliferam e se diferenciam, levando a uma rede túbulo-alveolar altamente ramificada. Concomitantemente, as células epiteliais mamárias (SAMU) tornam-se polarizado e capaz de sintetizar e secretar produtos lácteos. MECs organizar-se em numerosas estruturas alveolares (acini) que são cercados por células mioepiteliais contráteis e incorporadas em um estroma composto por tecidos conectivos e adiposos, vasos sanguíneos e terminações nervosas (Figura 1B, a gravidez). Além disso, o lado basal de MECs está em estreito contato com a membrana basal (matriz extracelular), e as interações entre estas duas entidades com força regular tanto a função morfogênese e secretora do mammary epitélio 11-13. Todos estes processos dependem da acção de várias sugestões ambientais, das quais as mais importantes são hormones14, factores parácrinos e a matriz extracelular. Por exemplo, a progesterona induz extensa alveologenesis 15 e que, em combinação com a prolactina (PRL) lateral ramificando-16,17, promove a diferenciação e mantém dos alvéolos. Em adição aos esteróides e PRL18, citoquinas e as vias de sinalização associadas ao desenvolvimento (Wnt e Notch vias de sinalização) também estão envolvidos em compromisso e desenvolvimento mamário linhagem 19-21. No final da gravidez, as MECs luminais começar a produzir um leite rico em proteína conhecida como colostro no lúmen dos alvéolos. Além disso, a progesterona actua sobre a permeabilidade epitelial e uma vez que as junções apertadas são ainda aberta, colostro também é encontrado na corrente sanguínea da mãe. Após o parto, a Mammary epitélio ocupa quase todo o volume da glândula mamaria e é altamente organizada (Figura 2, epitélio mamário). Unidades de produção de leite, nomeadamente alvéolos (Figura 2, alvéolo), são formadas por uma monocamada de células epiteliais mamarias secretórias polarizados (MESCs), com a sua membrana plasmática apical que delimita o lúmen. Alvéolos organizar-se em lóbulos que são agrupados em lobos ligados a condutas que drenam o leite para o meio exterior (Figura 2, lobo). Lactação ocorre, isto é., MESCs começar a secretar grandes quantidades de leite, provocados principalmente pela queda de hormonas da placenta (principalmente progesterona) (Figura 1B, lactação). Genes de proteínas do leite são activados de um curso de tempo variando temporais definido desde a gravidez até lactação 9,22,23, principalmente em resposta a pituitária PRL libertado no momento da sucção. Concomitantemente, os contactos entre MESCs e da matriz extracelular tanto estimular a proteína do leite synthesis através de sinais que são mediadas através das interacções entre integrinas celulares e laminina 24,25, e suprimir a apoptose em MESCs 26,27. Estas vias de sinalização como resultado a activação de promotores de genes de proteínas do leite 28 através da activação da transcrição de factores específicos 29. Contatos célula-célula também são importantes para alguns aspectos de diferenciação, incluindo o estabelecimento de polaridade apical ea secreção vectorial de produtos lácteos. Tight junctions fechar rapidamente após o início da lactação e MESCs finamente orquestrar a absorção de moléculas a partir do sangue, bem como a síntese, o transporte e a secreção de componentes de leite, em resposta aos requisitos nutricionais dos recém-nascidos. No momento da sucção, a contracção das células mioepiteliais que rodeiam os alvéolos ocorre em resposta à oxitocina e leva a ejecção de leite através das condutas e para dentro do mamilo. O leite é um fluido que contém complexo de proteínas (principalmentecaseínas), açúcares (principalmente lactose), lípidos e minerais, assim como moléculas bioactivas, tais como imunoglobulinas A (IgA), factores de crescimento e hormonas. As caseínas são sintetizados, montados em estruturas supramoleculares, ou seja micelas de caseína, transportados ao longo da via secretora, e, em seguida, libertada por exocitose, isto é, a fusão de vesículas secretoras contendo caseína (SVS) com a membrana plasmática apical de MESC (Figura 2). Tráfego intracelular depende de trocas materiais entre os compartimentos membranosos e envolve Solúvel N-etilmaleimida Sensitive Fusion (NSF) Protein Anexo (SNAP) Receptor (SNARE) 30,31. A família de proteínas SNARE é subdividida em SNAREs vesiculares (v-SNAREs), presentes na membrana da vesícula, e laços alvo (t-armadilhas), localizadas nas membranas alvo. Ao fechar através dos seus domínios em espiral espiralada, V- e t-SNARE montar para formar um complexo de feixe de quatro hélices altamente estável, referido como thcomplexo e SNARE. Este complexo promove a fusão de duas camadas duplas lipídicas opostos gradualmente trazê-los em estreita proximidade 30,32. Em seguida, os complexos SNARE são dissociados pelo triphosphatase adenosina NSF e suas proteínas SNAP e SNARE proteína adaptador são reciclados de volta ao seu compartimento de origem 33. Interessantemente, cada proteína SNARE reside predominantemente em compartimentos celulares distintas e SNARE emparelhamento pode contribuir para a especificidade de eventos intracelulares de fusão 34. Estudos anteriores sugerem que, pelo menos, Protein 23 (SNAP23) e vesículas com Associated proteína de membrana 8 (VAMP8), e syntaxins (STX) sinaptosomal Associada -7 e -12 desempenhar um papel na caseína exocitose 35,36. Estas proteínas também foram encontrados em associação com a fracção lipídica de leite, isto é, gordura de leite (MFG glóbulos) 37. O modelo predominante atual postula que gotículas lipídicas citoplasmáticas (CLDS) são formados pela acumulação de neutro lIPIDs (principalmente triacilgliceróis e ésteres de esteróis) e colesterol derivado da dieta materna entre os dois folhetos do retículo endoplasmático (ER) da membrana 38-41. Grandes CLD são formados, pelo menos em parte, pela fusão de CLD menor enquanto está a ser transportado para o lado apical de MESCs onde eles são libertados como MFG (1-10 um de diâmetro) por germinação, sendo envolta pela membrana plasmática apical MESC 40-42. Aleitamento cessa após os filhotes são desmamados e os MESCs morrem progressivamente por apoptose, levando à regressão do tecido mamário volta a um estado puberal (Figura 1B, involução). Imunofluorescência (IF) é um método de laboratório analítico comum usado em quase todos os aspectos da biologia, tanto em pesquisas como em diagnósticos clínicos. SE técnicas podem ser realizadas em secções de tecido (imuno-histoquímica, IHC) ou imunocitoquímica (ICC), amostras de células. Esta poderosa abordagem baseia-se na utilização de fluorescent-anticorpos marcados que se ligam especificamente (directa ou indirectamente) para o antigénio de interesse, permitindo assim a visualização da sua distribuição nos tecidos através de microscopia de fluorescência. Principalmente sinais de fluorescência dependem da qualidade e concentração dos anticorpos e tratamento adequado da amostra. Um protocolo simples de imunofluorescência indirecta (IIF) é apresentada para detectar produtos de leite (caseínas e MFG) e proteínas envolvidas na secreção do produto de leite (butyrophilin (btn1), proteínas SNARE) em secções congeladas de tecido mamário de ratinho (Figura 3). Embora este protocolo fornece uma visão completa IHC, que vão desde coleta de tecido para a imagem de pós-tratamento, crítica e passos opcionais, bem como algumas recomendações técnicas também são apresentados e discutidos.

<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

A imunofluorescência indirecta é utilizado para detectar e localizar as proteínas de interesse num tecido. O protocolo aqui apresentado descreve um método completo e simples para a detecção imunológica de proteínas, o rato lactantes glândula mamária ser tomado como um exemplo. Um protocolo para a preparação das amostras de tecidos, especialmente em relação a dissecção da glândula mamária do mouse, fixação do tecido e corte de tecido congelado, são detalhados. Um protocolo padrão para executar imunofluorescência indireta, incluindo uma etapa de recuperação antigênica opcional, também é apresentada. A observação de as secções de tecido etiquetados, bem como a aquisição de imagem e os pós-tratamentos também são apresentados. Este procedimento dá uma visão geral completa, desde a coleta de tecido animal para a localização celular de uma proteína. Embora este método geral pode ser aplicada a outras amostras de tecido, deve ser adaptada a cada tecido / anticorpo primário par estudado.

A glândula mamária é uma glândula subcutânea localizada ao longo da estrutura de ambos ventral do tórax e do abdômen em roedores. A localização dos cinco pares de glândulas do rato durante a gestação é mostrado na Figura 4. A morfologia da glândula mamária muda dramaticamente durante o seu desenvolvimento, reflectindo modificações funcionais necessárias para preparar a plena lactação (Figura 1B). Em animais virgens ou nulíparas, a glân...

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

O protocolo de imunofluorescência indirecta descrito neste artigo permite a detecção e a localização de proteínas na glândula mamaria do rato. Um método completo é dada para preparar amostras da glândula mamaria, a efectuar imuno-histoquímica, para as secções de tecido imagem por microscopia de fluorescência, e para reconstruir imagens.

Imunofluorescência Indireta em cortes congelados de Rato glândula mamária

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.

Imuno-histoquímica em parafina da epiderme do rato utilizando anticorpos fluorescentes

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

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.

Rato células epiteliais mamárias forma Mammospheres durante a diferenciação lactogênica

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.

Alterações na morfologia da glândula mamária e risco de câncer de mama em ratos

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.

Seções incluído em parafina e congelados de Drosophila Músculos Adulto

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 de Células-Tronco do rato das Glândulas Salivares

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.

Avaliação do desenvolvimento da glândula mamária e Função em modelos de ratos

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.

Profiling única célula transcricional dos cardiomiócitos de rato adulto

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.

Injeção Intraductal para localizada Drug Delivery ao Rato glândula mamária

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.

Efeito do Masculino Acessório Gland produtos em postura de ovos em gastrópode

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 de glândulas mamárias de Mouse intacto, todo para a análise da expressão de matriz extracelular e morfologia da glândula

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 ...