Name: preparation of murine submandibular salivary gland for upright intravital microscopy
Uploaded: 2018-05-07T12:30:00
Description: Watch this Scientific Journal Video about Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy at JoVE.com

Dette arbejde blev finansieret af schweiziske nationale Foundation (SNF) project grant 31003A_135649, 31003A_153457 og 31003A_172994 (til JVS) og Leopoldina fellowship LPDER 2011-16 (til BS). Dette arbejde nydt godt af optiske opsætninger af "Mikroskopi Imaging Center" (MIC) af Berns universitet.

Alle dyr arbejde er godkendt af den kantonale Udvalget for dyr eksperimenter og gennemføres i henhold til føderale retningslinjer.
1. bedøver musen
<ol><li>Bære personlige værnemidler, herunder laboratoriekittel og handsker.</li>
	<li>Bland ketamin, xylazin og saltvand til en fungerende koncentration af 20 mg/mL og 1 mg/mL, henholdsvis. Injicere arbejder stock intraperitoneal (i.p.) på 8-10 µL/g af musen. Placer musen tilbage ind i buret. Bemærk: Denne protokol er blevet testet fo...

<ol>
	<li>Pakurar, A. S., Bigbee, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digestive+System.">Digestive System.</a> Digital Histology. , 101-121 (2005).</li><li>Carpenter, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+Saliva+in+the+Oral+Processing+of+Food.">Role of Saliva in the Oral Processing of Food.</a> Food Oral Processing. , 45-60 (2012).</li><li>Masedunskas, A., Weigert, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+two-photon+microscopy+for+studying+the+uptake+and+trafficking+of+fluorescently+conjugated+molecules+in+live+rodents.">Intravital two-photon microscopy for studying the uptake and trafficking of fluorescently conjugated molecules in live rodents.</a> Traffic. 9 (10), 1801-1810 (2008).</li><li>Amano, O., Mizobe, K., Bando, Y., Sakiyama, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+histology+of+rodent+and+human+major+salivary+glands.">Anatomy and histology of rodent and human major salivary glands.</a> Acta Histochem Cytochem. 45 (5), 241-250 (2012).</li><li>Sequeira, S. 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Denne protokol tilbyder en ligetil tilgang til i vivo billeddannelse af murine submandibulære og sublinguale spytkirtler ved hjælp af oprejst ikke-lineære mikroskopi ofte bruges inden for immunologi. Metoden kan være tilpasset til billeddannelse af andre eksokrine kirtler i regionen hoved og hals. For eksempel, har vores lab udført billeddannelse af lacrimal kirtel på en tilsvarende måde (ikke vist).
At fjerne bindevæv omkring SMG er den mest afgørende skridt i denne protokol...

Spyt er udskilles af eksokrine kirtler at smøre mad, beskytte slimhindeinfektioner af mundtlige tarmkanalen og levere fordøjelsesenzymer samt antimikrobielle stoffer1,2. Foruden mindre spytkirtlerne afbrudt i den mundtlige submucosa, er der tre bilaterale sæt af store kirtler identificeret som parotideale, sublinguale og submandibulære, efter deres placering1,2. Pyramide-formet epitelceller, organiseret i kolbe-formet sække (acini) eller demilunes, der er omgivet af myoepithelial celler og en basalmembran, udskiller komponenterne serøs og slimhinderne af spyt1. Den smalle luminale plads i acini afløb i indtrængere kanaler, der forener i tværstribede kanalerne, indtil de endelig kommer ind i en enkelt ekskretionsorganerne kanal1. De vigtigste ekskretionsorganerne kanalen af SMG kaldes Whartons kanalen (WD) og åbner i sublinguale caruncle3,4. SMG epitelial rum udgør derfor et stærkt arborized struktur med mangfoldige terminal slutpunkter, der ligner et bundt af druer1,5,6. SMG interstitium er sammensat af blod og lymfe-fartøjer indlejret i bindevæv7 indeholdende parasympatiske nerver8 og ekstracellulære matrix5. Normale menneskelige og gnaver spytkirtler også indeholde T celler, makrofager og dendritiske celler9, samt plasmaceller, der udskiller Immunoglobulin en (IgA) i spyt9,10. På grund af sit mangeartede funktioner i sundhed og sygdom er SMG et emne af interesse for mange områder inden for biologisk forskning, herunder tandpleje4, immunologi11, onkologi12, fysiologi8og cellebiologi 3.
Billeddannelse af dynamiske cellulære processer og interaktioner er et kraftfuldt værktøj i biologiske forskning13,14. Udviklingen af dybe væv imaging og innovationer inmicroscopes baseret på ikke-lineær optik (NLO), som er afhængige af spredning eller absorption af flere fotoner af prøven, har lov til at undersøge direkte cellulære processer i komplekse væv13 ,15. Absorption af flere fotoner indebærer levering af den samlede excitation energi af lav energi fotoner, som begrænser fluorophore excitation til brændplanet og dermed giver mulighed for dybere væv penetration med reduceret solskader og støj fra ud af fokus excitation13,15. Dette princip er ansat ved to-foton mikroskopi (2 PM) og giver mulighed for billeddannelse af fluorescerende modellerne i dybet af op til 1 mm15,16. Mens kommercielt tilgængelige 2 PM opsætninger er blevet brugervenlige og pålidelige, er den store udfordring for intravital billeddannelse omhyggeligt udsætte og stabilisere målorgan bedøvede mus, især for billeddannelse af time lapse serie. Flere metoder til digital drift korrektion efter dataopsamling har været udgivet17,18 , og vi for nylig udviklet "VivoFollow", en automatisk korrektion system, som modvirker langsom væv afdrift i realtid ved hjælp af en edb trin19. Men det er stadig kritisk for høj kvalitet imaging for at minimere væv bevægelse, især hurtige bevægelser skyldes vejrtrækning eller puls19. Forberedelse og stabilisering procedurer er blevet udgivet til flere organer, herunder rygmarven20, leveren21, hud22, lunge23og lymfeknude24. Derudover har modeller for rotte spytkirtel imaging været udviklet3,25 og yderligere raffineres for høj opløsning intravital billeddannelse af murine SMG skræddersyet til en inverteret mikroskop setup26, 27 , 28.
Her præsenterer vi en praktisk og fleksibel protokol for intravital billeddannelse af murine SMG bruger oprejst nonlinear mikroskopi, som er almindeligt anvendt til intravital imaging inden for immunologi. Med henblik herpå ændrede vi et bredt ansat immobilisering fase bruges til popliteal lymfeknude præparater.

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			<td>Narketan 10 %&#160; (Ketamine) 20ml (100 mg/ml)</td>
			<td>Vetoquinol</td>
			<td>3605877535982</td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun 2% (Xylazine) 25 ml (20 mg/ml)</td>
			<td>Bayer</td>
			<td>680538150144</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline NaCl 0.9%</td>
			<td>B. Braun</td>
			<td>3535789</td>
			<td></td>
		</tr>
		<tr>
			<td>Prequillan 1% (Acepromazine) 10 ml (10 mg/ml)</td>
			<td>Fatro</td>
			<td>6805671900029</td>
			<td></td>
		</tr>
		<tr>
			<td>Electric shaver</td>
			<td>Wahl</td>
			<td>9818L</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Hair removal cream</td>
			<td>Veet</td>
			<td>4002448090656</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)</td>
			<td>Durapore (3M)</td>
			<td>1538-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Durapore Surgical tape (2.5 cm x 9.1 m and 1.25 cm x 9.1 m)</td>
			<td>Durapore (3M)</td>
			<td>1538-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Super glue Ultra gel, instantaneous glue</td>
			<td>Pattex, Henkel</td>
			<td>4015000415040</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass slides 20 mm and 22 mm</td>
			<td>Menzel-Gl&#228;ser</td>
			<td>631-1343/ 631-1344</td>
			<td></td>
		</tr>
		<tr>
			<td>Grease for laboratories 60 g glisseal N</td>
			<td>Borer (VWR supplier)</td>
			<td>DECO514215.00-CA15</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Fine Science Tools (F.S.T )</td>
			<td>14090-09</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Fine Forceps</td>
			<td>Fine Science Tools (F.S.T )</td>
			<td>11252-20</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td>Migros</td>
			<td>617027988254</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Gauze Gazin 5 x 5 cm</td>
			<td>Lohmann and Rauscher</td>
			<td>18500</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Leica</td>
			<td>MZ16</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>Texas Red dextran 70kDa</td>
			<td>&#160;Molecular Probes</td>
			<td>D1864</td>
			<td></td>
		</tr>
		<tr>
			<td>Cascade Blue dextran 10kDa</td>
			<td>invitrogen</td>
			<td>D1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Two-photon system</td>
			<td>LaVision Biotec</td>
			<td>TrimScope I and II</td>
			<td>or similar</td>
		</tr>
		<tr>
			<td>XLUMPLANFL 20x/0.95 W objective</td>
			<td>Olympus</td>
			<td>n/a</td>
			<td>or other water immersion objective&#160;</td>
		</tr>
		<tr>
			<td>Digital thermometer</td>
			<td>Fluke</td>
			<td>95969077651</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of murine submandibular salivary gland for upright intravital microscopy

De submandibulære spytkirtel (SMG) er en af de tre store spytkirtler, og er af interesse for mange forskellige områder af den biologiske forskning, herunder cellebiologi, onkologi, tandpleje og Immunologi. SMG er en eksokrine kirtel består af sekretoriske epitelceller, myofibroblasts, endotel celler, nerver og ekstracellulære matrix. Dynamisk cellulære processer i rotter og mus SMG har tidligere været afbildet, for det meste bruger omvendt multi photon mikroskop systemer. Her beskriver vi en enkel protokol for kirurgisk forberedelse og stabilisering af den murine SMG i bedøvede mus for i vivo billeddannelse med oprejst multi photon mikroskop systemer. Vi præsentere repræsentativt intravital billede sæt af endogene og overført adoptively fluorescerende celler, herunder mærkning af blodkar eller spyt kanalerne og andet harmonisk generation til at visualisere fibrillar kollagen. I sum, vores protokol giver mulighed for kirurgisk forberedelse af mus spytkirtlerne i opretstående mikroskopi systemer, som er almindeligt anvendt til intravital imaging inden for immunologi.

Denne protokol tillader billeddannelse af næsten den hele dorsale eller ventrale side af SMG. Synsfeltet omfatter typisk også den sublinguale spytkirtel, der adskiller sig lidt fra SMG i cellulære sammensætning4. Begge kirtler er indkapslet af fibrillar kollagen og opdelt i fliger. De fleste 2 PM systemer kan producere en etiket-gratis billede af fibrillar kollagen ved at måle 2nd harmoniske signal, men normalt fluorescerende molekyler er nødvendig...

Watch this Scientific Journal Video about Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy at JoVE.com

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

Forberedelse af Murine submandibulære spytkirtel til opretstående Intravital mikroskopi

Vi beskriver en protokol for at kirurgisk afsløre og stabilisere den murine submandibulære spytkirtel for intravital billeddannelse ved hjælp af opretstående intravital mikroskopi. Denne protokol er let at tilpasse til andre eksokrine kirtler af hoved og hals region af mus og andre små gnavere.

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy of the Inguinal Lymph Node

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Intravital Imaging af Mouse Thymus med 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

Intravital Mikroskopi af milten: Kvantitativ analyse af Parasite mobilitet og Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Spyt, spytkirtel, og hæmolymfe Collection fra Ixodes scapularis Flåter

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Stærkt Løst Intravital Striped-belysning mikroskopi af kimcentre

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital Microscopy Imaging af Liver efter<em&gt; Leishmania</em&gt; Infektion: En Vurdering af Nedsat Hæmodynamik

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Long Term Intravital Multiphoton Microscopy Imaging of Immune Cells in Healthy and Diseased Liver Using CXCR6.Gfp Reporter Mice

Long Term Intravital multiphoton Microscopy Imaging af immunceller i rask som syg Lever Brug CXCR6.Gfp Reporter Mus

Liver inflammation as a response to injury is a highly dynamic process involving the infiltration of distinct subtypes of leukocytes including monocytes, ...

A Method for the Measurement of Salivary Gland Function in Mice

En metode til måling af spytkirtel funktion i mus

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

Imaging celle interaktion i trakeal slimhinde under Influenza virusinfektion ved hjælp af to-foton Intravital mikroskopi

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...