Name: Microscopia subcellulare intravitale della ghiandola mammaria (Video) | JoVE Journal
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Duration: 4 min 14 s
Description: Impara la microscopia confocale e multifotone per l'imaging intravitale della ghiandola mammaria in topo lattante. Questo metodo consente di visualizzare in tempo reale la dinamica dei lipidi e l'architettura cellulare con alta risoluzione.

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</ol>

<table><tbody><tr><td>488 laser</td><td>Melles-Griot-CW</td><td></td><td>&nbsp; laser 50 mW</td></tr><tr><td>60x PLAPON obiettivo a immersione in olio (NA 1.42)</td><td>Olympus</td><td>1-U2B933</td><td>Lente Microscopio confocale</td></tr><tr><td>633 laser</td><td>Melles-Griot-CW</td><td></td> <td>He-Ne 12 mW</td></tr><tr><td>Obiettivo 63x (NA 1.40, HC PL APO CS2)</td><td>Leica</td><td>11506350</td><td>Lens Microscopio a due fotoni</td></tr><tr><td>BA 410-460 nm</td><td>Chroma</td><td>-</td><td>Filtro passa-banda</td></tr><tr><td>BA 495-540 nm</td><td></td> <td>Filtro passa-banda</td><td>Chroma</td></tr> <tr><td>BA 505-605 nm</td> <td>Filtro passa-banda</td><td>Chroma</td><td></td></tr> <tr><td>BA 655-755 nm</td> <td>Filtro passa-banda</td><td>Chroma</td><td></td></tr> <tr><td>Boro-dipirrometano (BODIPY) 665/676</td><td>Thermo Fisher Scientific</td><td>B3932</td><td>Sensore di perossiazione lipidica</td></tr><tr><td>Carbomer-940</td><td>Snowdrift Farm</td><td>Catetere in</td><td>gel</td>739601480651</tr><tr><td></td><td>Terumo</td><td>SV27EL</td><td>Set di infusione alati&nbsp;</td></tr><tr><td>Cauterizzatore&nbsp;</td><td>Braintree Scientific, Inc</td><td>GEM 5917</td><td>Sistema di cauterizzazione</td></tr><tr><td>CMV-Cre topo&nbsp;</td><td>Jackson lab</td><td>006054</td><td>Mouse line</td></tr><tr><td>Coverslip</td><td>Bioptechs-30mm</td><td></td> <td>diametro coverlip per microscopio invertito</td></tr><tr><td>Curity 4x4 pollici multiuso spugna garza</td><td>Covidien</td><td>9024</td><td>Sponge</td></tr><tr><td>EGFPcyto mouse</td><td>Jackson lab</td><td>003291</td><td>Mouse line</td></tr><tr><td>Fiji/ImageJ software</td><td>Open</td> <td>source-Free</td> software <td></td></tr><tr><td>Pinze fini</td><td>Braintree Scientific, Inc</td><td>FC003 8</td><td>Pinze per tessuti</td></tr><tr><td>Microscopio Fluoview 1000</td><td>Microscopio confocale</td><td>Olympus</td><td>FV1000</td></tr><tr><td>Software FluoView Microscopio</td> <td>confocale Olympus e microscopio a due fotoni</td><td></td><td></td></tr> <tr><td>Rasoio elettrico portatile</td><td>Braintree Scientific, Inc</td><td>CLP-8786-451A</td><td>Clipper senza fili</td></tr><tr><td>Termoforo</td><td>Braintree Scientific, Inc</td><td>DPIP</td><td>Pad termico per animali</td></tr><tr><td>Rivelatori HyD</td><td>Leica-Leica</td><td></td> <td>4Tune rivelatore spettrale</td></tr><tr><td>Software Imaris</td><td>Bitplane / Oxford instruments-Strumento</td><td></td> <td>software commerciale</td></tr><tr><td>Ingisht X3 laser sintonizzabile</td><td>Spectra Physics</td><td>Insight X3</td><td>Tunable Pulse-Laser</td></tr><tr><td>Isoflurane</td><td>VetOne</td><td>13985-046-40</td><td>Ketamina anestetica</td></tr><tr><td>&nbsp;</td><td>VetOne</td><td>13985-702-10</td><td>Anestetico</td></tr><tr><td>LAS X Software</td><td></td> Leica-Two-Photon <td></td><td>microscopio strumento software</td></tr><tr><td>Mai-Tai laser sintonizzabile</td><td>Spectra Fisica Mai-Tai</td><td></td><td>Laser</td></tr><tr><td>MetaMorph</td><td>Molecular Devices-Strumento</td><td></td> <td>software commerciale</td></tr><tr><td>Monodansilpentano AUTODOT</td><td>Abcepta</td><td>Sm1000a</td><td>Colorante goccioline lipidiche</td></tr><tr><td>MPE-RS microscopio</td><td>Olympus</td><td>IX70</td><td>Microscopio a due fotoni</td></tr><tr><td>mouse mT/mG</td><td>lab</td><td>Jackson 007676</td><td>Linea di topi</td></tr><tr><td>Riscaldatore dell'obiettivo</td><td>Bioptechs</td><td>150819</td><td>Riscaldatore dell'obiettivo per microscopi confocali e a due fotoni</td></tr><tr><td>Camera di respirazione satura di ossigeno</td><td>Patterson Scientific</td><td>78933385, SAS3 ed EVAC4</td><td>Sistema di anestesia ed evacuazione dei gas...</td></tr><tr><td>Parafilm</td><td>Heathrow Scientific</td><td>HS234526B</td> Rivelatore PMT <td>a film termoplastico semitrasparente, flessibile</td></tr><tr><td></td><td>Olympus-Descanned</td><td></td><td>&nbsp; &nbsp; rivelatori</td></tr><tr><td>PMT Rivelatore</td><td>LSM-Technology</td><td>Rivelatori non descanneti</td><td>costruiti su misura</td></tr><tr><td>Pompa</td><td>Harvard Apparatus</td><td>703602, 704402</td><td>Iniettore e controller di nanoacari</td></tr><tr>Qualità<td>salina</td><td>Biologico</td><td>114-055-721EA</td><td>Soluzione salina normale</td></tr><tr><td>Forbici affilate con estremità smussate</td><td>Braintree Scientific, Inc</td><td>SCT-S 508</td><td>Forbici</td></tr> <tr><td>chirurgicheSiringa</td><td>Covidien</td><td>22-257-150</td><td>Siringa tubercolina da 1 ml</td></tr><tr><td>TCS SP8 Microscopio spettrale da immersione</td><td>Leica</td><td>SP8</td><td>Microscopio a due</td></tr><tr><td>fotoni Pinzette&nbsp;</td><td>Braintree Scientific, Inc</td><td>FC032</td><td>Pinze per tessuti</td></tr><tr><td>Xilazina&nbsp;</td><td>VetOne</td><td>13985-704-10</td><td>Anestetico</td></tr></tbody></table>

microscopia subcellulare intravitale della ghiandola mammaria

Intravital Subcellular Microscopy of the Mammary Gland

Il latte è secreto da cellule epiteliali alveolari polarizzate, che si differenziano durante la gravidanza dalle gemme di un esteso albero duttulare26 (Figura 2A). I precursori per la sintesi del latte vengono assimilati attraverso le membrane basali/laterali e i prodotti completi vengono secreti attraverso la superficie apicale in uno "spazio del latte" centrale. Il lato basale di ciascun alveolo è coperto da una serie stellata di cellule mioepiteliali (<strong class...

Microscopia subcellulare intravitale della ghiandola mammaria

The mammary gland constitutes a model par excellence for investigating epithelial functions, including tissue remodeling, cell polarity, and secretory ...

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Time-lapse imaging viene utilizzato per valutare il comportamento di primarie preneoplastiche cellule epiteliali mammarie derivate da modelli murini geneticamente modificati di rischio di cancro al seno per determinare se ci sono correlazioni tra i parametri specifici di comportamento e distinte lesioni genetiche.

Time-lapse imaging di primaria preneoplastiche cellule epiteliali mammarie Derivato da modelli murini geneticamente modificati di cancro al seno

La microscopia intravitale è un potente strumento che permette l'imaging vari processi biologici di animali vivi. In questo articolo, presentiamo un metodo dettagliato per l'imaging la dinamica di strutture subcellulari, come granuli secretori, nelle ghiandole salivari di topi vivi.

Intravitale Microscopia per le strutture Imaging subcellulari in topi vivi Esprimere proteine ​​fluorescenti

Un protocollo per la consegna intraduttale non invasiva di reagenti acquosi alla ghiandola mammaria topo è descritto. Il metodo si avvale di iniezione localizzato nei capezzoli delle ghiandole mammarie destinati dotti mammari in particolare. Questa tecnica è adattabile per una varietà di composti compresi siRNA, agenti chemioterapici e piccole molecole.

Iniezione intraduttale per Localizzato Drug Delivery per il mouse ghiandola mammaria

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'immagine delle sezioni di tessuto al microscopio a fluorescenza, e di ricostruire le immagini.

Immunofluorescenza indiretta su sezioni congelate di mouse ghiandola mammaria

Questo protocollo descrive l'uso della microscopia multiphoton effettuare prolungato time-lapse imaging di interazioni multicellulari in tempo reale, in vivo a risoluzione singola cellula.

Esteso Time-lapse Imaging intravitale del Real-time multicellulare Dynamics nel microambiente tumorale

Qui, descriviamo una tecnica per la somministrazione localizzata di reagenti alla ghiandola mammaria del coniglio tramite un'iniezione intraduttale. Inoltre, descriviamo un protocollo per la visualizzazione e la conferma della somministrazione mediante imaging ecografico ad alta risoluzione degli agenti di contrasto.

Consegna intraduttale al Coniglio ghiandola mammaria

Qui, presentiamo un protocollo per l'isolamento del mouse intero, intatto ghiandole mammarie per studiare l'espressione di matrice extracellulare (ECM) e morfologia duttale. Mouse #4 ghiandole addominali sono state estratte da topi femmine nullipare 8-10 settimane vecchio, fissato in formalina neutra tamponata, sezionato e macchiato usando immunohistochemistry per proteine ECM.

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

Viene qui presentato un approccio per l'imaging intravitale a lungo termine utilizzando finestre in silicone otticamente chiare che possono essere incollate direttamente al tessuto / organo di interesse e alla pelle. Queste finestre sono più economiche e versatili di altre attualmente utilizzate sul campo e l'inserimento chirurgico provoca un'infiammazione e un disagio limitati agli animali.

Imaging intravitale longitudinale tramite finestre in silicone trasparente

Questo protocollo descrive una nuova finestra di imaging mammario con un coperchio sostituibile (R.MIW). La microscopia intravitale dopo l'impianto del R.MIW consente l'imaging longitudinale e di più giorni della ghiandola mammaria sana e malata con una risoluzione cellulare durante le diverse fasi dello sviluppo.

Microscopia intravitale longitudinale utilizzando una finestra di imaging mammario con coperchio sostituibile

Il metodo di imaging intravitale qui descritto utilizza la seconda generazione armonica di collagene e la fluorescenza endogena dal cofattore metabolico NAD(P)H per segmentare in modo non invasivo un microambiente tumorale non etichettato in compartimenti tumorali, stromali e vascolari per l'analisi approfondita delle immagini intravitali 4D.

Un approccio di segmentazione senza etichette per l'imaging intravitale del microambiente del tumore mammario

Il presente protocollo descrive l'iniezione intraduttale di vettori virali attraverso la tettarella per fornire geni di interesse nelle cellule epiteliali mammarie.

In vivo Consegna genica nelle cellule epiteliali mammarie di topo attraverso l'iniezione intraduttale mammaria

Impara la microscopia confocale e multifotone per l'imaging intravitale della ghiandola mammaria in topo lattante. Questo metodo consente di visualizzare in tempo reale la dinamica dei lipidi e l'architettura cellulare con alta risoluzione.

Microscopia subcellulare intravitale della ghiandola mammaria

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