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

Forfatterne er taknemmelige for INRA MIMA2 imaging core facilitet (INRA, UMR1198, Jouy-en-Josas), og til de ansatte i IERP enhed (UE 0907, INRA, Jouy-en-Josas) til pasning af dyr og faciliteter. Vi vil også gerne takke IH Mather, MC Neville og S. Tooze for at give os meget nyttige antibodie.

CD1 mus blev avlet på INRA (UE0907 IERP, Jouy-en-Josas, Frankrig). Alle etiske aspekter af pasning af dyr i overensstemmelse med de relevante retningslinjer og licenskrav, der er fastsat af det franske landbrugsministerium. De anvendte procedurer blev godkendt af den lokale etiske komité (aftale 12/097 fra Comethea Jouy-en-Josas / AgroParisTech). 1. brystkirtel Prøvefremstilling <ol><li> Mus brystkirtel dissektion <ol><li> Aflive mus på dag 10 af amning ved cervikal dislokatio...

IHC er en relativt enkel og ligetil metode til eksperimentel lokalisere antigen i vævssnit, som først og fremmest på specifikke epitop-antistof-interaktioner. Selvom der anvendes et stort antal protokoller til at lokalisere en protein ved IIF, kernen i disse procedurer er næsten altid den samme. Men der er nogle kritiske aspekter, der kan stærkt påvirke resultatet og skal derfor optimeret til hver enkelt IHC undersøgelse. Den mest udfordrende aspekt af denne fremgangsmåde er at bestemme den bedste eksperimentell...

Mælkekirtlen er en atypisk pattedyr exokrin orgel, hvis vigtigste funktion er at producere mælk til foder nyfødte. Udviklingen af ​​brystvæv forekommer hovedsageligt efter fødslen og er kendetegnet ved en unik fremgangsmåde, hvor epitel invaderer omgivende stroma. Dette væv undergår mange ændringer (vækst, differentiering og regression), især i løbet af voksenlivet, samtidig med variationer i reproduktiv status (figur 1). Ud over den samlede morfologi af vævet, andelene af forskellige celletyper samt deres arrangement inden mælkekirtlen dramatisk ændre under udviklingen 1-5. Under fosterstadiet, brystepiteliet stammer fra brystkirtler mælkeledninger, der defineres ved en svag fortykkelse og lagdeling af ektoderm, mellem for- og bagben på hver side af midterlinjen omkring embryonale dag 10.5 (E10.5) (figur 1A ).På E11.5, mælkeledningen bryder op i individuelle placodes, som er symmetrisk placeret langs bryst-mælkeledning på reproducerbare steder, og den omgivende mesenkym begynder at kondensere. De placodes begynder at synke dybere ind i dermis og brystkirtler mesenchyme organiserer i koncentriske lag omkring mamma opløbet (E12.5-E14.5). Pr E15.5, brystepiteliet, begynder at proliferere og langstrakte for at danne den primære spire, der skubber gennem mamma mesenkym mod fedtpuden. Den primære spire udvikler et hult lumen med en åbning til huden, kendetegnet ved dannelsen af ​​brystvorten kappe. På E18.5, har forlængede kanal vokset til fedtpuden og har forgrenet i en lille arborized gangsystemet omfattet i fedtpuden. Udvikling er hovedsagelig anholdt og den rudimentære brystkirtel forbliver morphogenetically hvile indtil puberteten. I den mandlige foster, aktiveringen af ​​androgen receptorer fører til degeneration af knopper, der forsvinderved E15.5. Pr E18, mammary udvikling ophører indtil puberteten 6-9. Ved fødslen, brystkirtlen huser en rudimentær gangsystemet, der forlænger og grene langsomt (isometrisk vækst). Ved starten af ​​puberteten, sfæriske strukturer placeret ved spidserne af kanalerne kaldes afslutningsenden knopper (TEBS), er dannet af et ydre lag af cap celler og et flerlaget indre kerne af celler (kropsceller). Disse strukturer er meget proliferativ og infiltrere det omgivende stromavæv som reaktion på hormonale signaler. Spredning i TEBS resulterer i duktalt forlængelse, kombineret med forgrening morfogenese. Denne proces fører til etablering af en grundlæggende epitelial arborized netværk stammer fra brystvorten (figur 1B, pubertet). På ~ 10-12 uger efter fødslen, når epitel har invaderet hele fedtpude, dens ekspansion stopper og TEBS forsvinde. Ductal udvikling gennemgår derefter dynamiske ændringer, dvs. successive spredning og regression af epitelceller ifølge Østralperioder 10 (figur 1B, voksne). Fra starten af ​​svangerskabet, brystvævet undergår vigtige vækst og morfologiske ændringer for at forberede amning. Brystepiteliet udførligt formering og differentiering, hvilket fører til en stærkt forgrenet tubulo-alveolær netværk. Samtidig hermed mammaepitelceller (MEC&#39;er) bliver polariseret og i stand til at syntetisere og udskille mejeriprodukter. MEC&#39;er organiserer i talrige alveolære strukturer (acini), der er omgivet af kontraktile myoepitelcellerne og indarbejdet i et stroma består af bindevæv og fedtvæv, blodkar og nerveender (figur 1B, graviditet). Desuden den basale side af MEC&#39;er er i tæt kontakt med basalmembranen (ekstracellulær matrix), og samspillet mellem disse to enheder streng regulering både morfogenese og sekretorisk funktion af mammary epitel 11-13. Alle disse processer er afhængige af virkningen af ​​forskellige miljømæssige tidskoder, hvoraf er de vigtigste hormones14, parakrine faktorer og den ekstracellulære matrix. For eksempel progesteron fremkalder omfattende side-forgrening 15 og alveologenesis, at der i kombination med prolactin (PRL) 16,17, fremmer og opretholder differentieringen af alveolerne. Ud over steroider og PRL18, cytokiner og signalveje i forbindelse med udvikling (Wnt og Notch signalveje) er også involveret i brystkirtler slægt engagement og udvikling 19-21. Ved slutningen af ​​graviditeten, begynder de luminale MEC&#39;er til frembringelse af et protein-rige mælk kendt som colostrum i lumen af ​​alveolerne. Desuden progesteron virker på epitel permeabilitet og da tight junctions stadig er åbne, er colostrum også fundet i moderens blod stream. Efter fødslen, det Mammary epitel optager næsten alle mælkekirtlen volumen og er stærkt organiseret (figur 2, brystepiteliet). Mælkeproducerende enheder, nemlig alveolerne (Figur 2, alveoler), er dannet af et monolag af polariserede brystepitelceller sekretoriske celler (MESCs), med deres apikale plasmamembran afgrænser hulrummet. Alveoler arrangere sig i lobules der er inddelt i lapper tilsluttet kanaler, der dræner mælken til ydersiden miljø (figur 2, lap). Amning opstår, dvs.., MESCs begynder at udskille rigelige mængder mælk, primært udløst af faldet i placenta hormoner (hovedsagelig progesteron) (figur 1B, amning). Milk-gener aktiveres i en defineret tidsmæssig tidsforløb i området fra graviditet til amning 9,22,23, hovedsageligt som svar på hypofyse PRL frigives på tidspunktet for diegivning. Samtidigt, kontakter mellem MESCs og den ekstracellulære matrix både stimulere mælkeprotein Synthesis gennem signaler, der medieres via samspillet mellem cellulære integriner og laminin 24,25, og undertrykker apoptose i MESCs 26,27. Disse signalveje resulterer i aktiveringen af mælkeprotein genpromotorer 28 gennem aktivering af specifikke transkriptionsfaktorer 29. Celle-celle kontakter er også vigtigt for nogle aspekter af differentiering, herunder etablering af apikale polaritet og vektorielle sekretion af mejeriprodukter. Tætte forbindelser hurtigt tæt efter begyndelsen af ​​laktationen og MESCs fint orkestrere optagelsen af ​​molekyler fra blodet samt syntese, transport og udskillelse af mælkebestanddele, som svar på de ernæringsmæssige krav hos nyfødte. På tidspunktet for diende, sammentrækning af myoepitelcellerne omgiver alveolerne sker som reaktion på oxytocin og fører til mælkenedlægningen gennem kanalerne og ind i brystvorten. Mælk er en kompleks væske, der indeholder proteiner (for det mestekaseiner), sukkerarter (hovedsagelig lactose), lipider og mineraler, samt bioaktive molekyler, såsom immunoglobuliner A (IgA), vækstfaktorer og hormoner. Kaseiner syntetiseres, samles i supramolekylære strukturer, nemlig caseinmiceller, transporteres langs den sekretoriske pathway, og derefter frigives ved exocytose, dvs. sammensmeltning af kasein-holdige sekretoriske vesikler (SV&#39;er) med den apikale plasmamembran Mesc (figur 2). Intracellulær trafik afhængig af materielle udvekslinger mellem hindeagtige rum og involverer Opløselige N-ethylmaleimid-Sensitive Fusion (NSF) Attachment Protein (SNAP) receptor (SNARE) 30,31. Snare proteiner familie er opdelt i vesikuløse Snares (v-Snares), der er til stede i vesikelmembranen og target snarer (t-Snares), lokaliseret på målet membraner. Ved zipping gennem deres dobbeltsnoet domæner, V- og T-snarer samles for at danne en meget stabil fire-helix bundt kompleks, benævnt the SNARE kompleks. Dette kompleks fremmer fusion af to modstående lipiddobbeltlag ved gradvist at bringe dem i tæt nærhed 30,32. Bagefter er SNARE komplekser dissocieret af NSF adenosintriphosphatase og dens adapter protein SNAP og SNARE proteiner recirkuleres tilbage til deres rum oprindelsesland 33. Interessant, hver SNARE protein overvejende er bosat i forskellige cellulære rum og SNARE parring kan bidrage til specificiteten af intracellulære fusion begivenheder 34. Tidligere undersøgelser tyder på, at mindst Synaptosomal-associeret protein 23 (SNAP23) og vesikelassocieret membranprotein 8 (VAMP8), og syntaxins (Stx) -7 og -12 spille en rolle i casein exocytose 35,36. Disse proteiner er også blevet fundet i forbindelse med lipidfraktion mælk, dvs. mælk fedtkugler (MFGs) 37. Den nuværende fremherskende model postulerer, at cytoplasmatiske lipiddråber (CLDs) dannes ved akkumulering af neutrale lipids (hovedsagelig triglycerider og sterolestere) og cholesterol afledt af moderens kost mellem de to foldere af det endoplasmatiske reticulum (ER) membran 38-41. Dannes store CLDs, i det mindste delvist, ved fusion af mindre CLDs medens de transporteres til den apikale side af MESCs hvor de er udgivet som MFGs (1-10 um i diameter) ved knopskydning, idet enwrapped af Mesc apikale plasmamembran 40-42. Amning ophører efter hvalpe er vænnet og MESCs gradvist dø ved apoptose, hvilket fører til regression af brystvævet tilbage til en pubertetsudvikling tilstand (figur 1B, involution). Immunofluorescens (IF) er en fælles analyselaboratorium metode, der anvendes i næsten alle aspekter af biologi, både i forskning og i klinisk diagnostik. HVIS teknikker kan udføres på vævssnit (immunhistokemi, IHC) eller celle (immuncytokemi ICC) prøver. Denne kraftfulde tilgang er baseret på anvendelsen af ​​fluorescent-mærkede antistoffer, der specifikt binder (direkte eller indirekte) til antigenet af interesse, hvilket tillader visualisering af dens vævsfordeling ved fluorescensmikroskopi. Fluorescenssignaler meste afhænger af kvaliteten og koncentration af antistofferne og korrekt håndtering af prøven. En simpel indirekte immunofluorescens (IIF) protokol præsenteres til påvisning af mejeriprodukter (kaseiner og MFGs) og proteiner involveret i mejeriprodukt sekretion (butyrophilin (BTN1), snare proteiner) på frosne snit af muse brystvæv (figur 3). Mens denne protokol giver et komplet IHC oversigt, der spænder fra væv samling til billedet efter behandling, kritisk og valgfrie trin samt nogle tekniske anbefalinger er også præsenteret og diskuteret.

<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

Indirekte immunofluorescens bruges til at detektere og lokalisere proteiner af interesse i et væv. Protokollen præsenteres her beskriver en fuldstændig og enkel fremgangsmåde til immunpåvisning af proteiner, idet musen diegivende brystkirtel taget som et eksempel. En protokol til fremstilling af vævsprøverne, især med hensyn til dissektion af musen brystkirtel, vævsfiksering og frosne væv sektionering, er detaljerede. En standardprotokol til at udføre indirekte immunfluorescens med et valgfrit antigen hentning trin, præsenteres også. Observationen af ​​de mærkede vævssnit samt billedoptagelse og post-behandlinger er også angivet. Denne procedure giver en fuld oversigt, fra indsamling af dyrevæv til cellulære lokalisering af et protein. Selv om denne generelle fremgangsmåde kan anvendes på andre vævsprøver, skal den tilpasses hvert væv / primært antistof par undersøgt.

Mælkekirtlen er en subkutan kirtel placeret langs den ventrale struktur både thorax og abdomen hos gnavere. Placeringen af de fem par kirtler i musen under svangerskabet er vist i figur 4. Morfologien af mælkekirtlen dramatisk ændrer under udviklingen, hvilket afspejler funktionelle ændringer, der kræves for at forberede fuld amning (figur 1B). I ubrugte eller barnløse dyr, brystkirtlen består af et tyndt forgrenet duktalt epitel indlejret i en tynd fede stroma, der...

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

Den indirekte immunofluorescens protokollen beskrevet i denne artikel giver mulighed for påvisning og lokalisering af proteiner i musen brystkirtel. En komplet fremgangsmåde er givet til at fremstille brystkirtel prøver, til at udføre immunohistokemi til afbildning af vævssnit ved hjælp af fluorescensmikroskopi, og at rekonstruere billederne.

Indirekte Immunofluorescens på frosne snit af Mouse brystkirtel

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.

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.

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.

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.

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.

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.

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 transkriptionel Profilering af Voksen Mouse cardiomyocytter

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.

Intraductal Injection til Lokaliseret Drug Delivery til Mouse mælkekirtlerne

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.

Effekt af Male tilbehør Gland Produkter på æglægning i havsnegle

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.

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

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...