Name: whole-mount immunofluorescence staining, confocal imaging and 3d reconstruction of the sinoatrial and atrioventricular node in the mouse
Uploaded: 2020-12-22T15:00:00
Description: Watch this Scientific Journal Video about Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse at JoVE.com

Dette arbejde blev støttet af China Scholarship Council (CSC, til R. Xia), det tyske center for kardiovaskulær forskning (DZHK; 81X2600255 til S. Clauss, 81Z0600206 til S. Kääb), Corona Foundation (S199/10079/2019 til S. Clauss), SFB 914 (projekt Z01 til H. Ishikawa-Ankerhold og S. Massberg og projekt A10 til C. Schulz), ERA-NET om hjerte-kar-sygdomme (ERA-CVD; 01KL1910 til S. Clauss) og Heinrich-and-Lotte-Mühlfenzl Stiftung (til S. Clauss). Bidragyderne havde ingen rolle i udarbejdelsen af manuskripter.

Dyrepleje og alle forsøgsforsøg blev udført i overensstemmelse med retningslinjerne fra Animal Care and Ethics Committee ved universitetet i München, og alle de procedurer, der blev udført på mus, blev godkendt af regeringen i Bayern, München, Tyskland (ROB-55.2-2532.Vet_02-16-106, ROB-55.2-2532.Vet_02-19-86). C57BL6/J mus blev købt fra Jackson Laboratory.
BEMÆRK: Figur 1 viser de instrumenter, der er nødvendige for eksperimentet...

<ol>
	<li>Clauss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+arrhythmia:+classic+electrophysiology+to+genetically+modified+large+animals.">Animal models of arrhythmia: classic electrophysiology to genetically modified large animals.</a> Nature Reviews Cardiology. 16 (8), 457-475 (2019).</li><li>Clauss, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+porcine+model+of+atrial+arrhythmogenicity+in+the+context+of+ischaemic+heart+failure.">Characterization of a porcine model of atrial arrhythmogenicity in the context of ischaemic heart failure.</a> PLoS One. 15 (5), 0232374 (2020).</li><li>Schuttler, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+Models+of+Atrial+Fibrillation.">Animal Models of Atrial Fibrillation.</a> Circulation Research. 127 (1), 91-110 (2020).</li><li>van Weerd, J. H., Christoffels, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+formation+and+function+of+the+cardiac+conduction+system.">The formation and function of the cardiac conduction system.</a> Development. 143 (2), 197-210 (2016).</li><li>Vogler, J., Breithardt, G., Eckardt, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bradyarrhythmias+and+Conduction+Blocks.">Bradyarrhythmias and Conduction Blocks.</a> Revista Espa&#241;ola de Cardiolog&#237;a (English Edition. 65 (7), 656-667 (2012).</li><li>Wen, Y., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+of+mouse+sinoatrial+node+and+its+expression+of+NF-160+and+HCN4.">Morphology of mouse sinoatrial node and its expression of NF-160 and HCN4.</a> International Journal of Clinical and Experimental Medicine. 8 (8), 13383-13387 (2015).</li><li>Verheijck, E. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+features+of+the+mouse+sinoatrial+node+in+relation+to+connexin+distribution.">Electrophysiological features of the mouse sinoatrial node in relation to connexin distribution.</a> Cardiovascular Research. 52 (1), 40-50 (2001).</li><li>Glukhov, A. V., Fedorov, V. V., Anderson, M. E., Mohler, P. J., Efimov, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+anatomy+of+the+murine+sinus+node:+high-resolution+optical+mapping+of+ankyrin-B+heterozygous+mice.">Functional anatomy of the murine sinus node: high-resolution optical mapping of ankyrin-B heterozygous mice.</a> American Journal of Physiology Heart and Circulatory Physiology. 299 (2), H482-H491 (2010).</li><li>Sillitoe, R. V., Hawkes, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+immunohistochemistry:+a+high-throughput+screen+for+patterning+defects+in+the+mouse+cerebellum.">Whole-mount immunohistochemistry: a high-throughput screen for patterning defects in the mouse cerebellum.</a> Journal of Histochemistry and Cytochemistry. 50 (2), 235-244 (2002).</li><li>Hulsmans, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+Facilitate+Electrical+Conduction+in+the+Heart.">Macrophages Facilitate Electrical Conduction in the Heart.</a> Cell. 169 (3), 510-522 (2017).</li><li>Liu, J., Dobrzynski, H., Yanni, J., Boyett, M. R., Lei, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organisation+of+the+mouse+sinoatrial+node:+structure+and+expression+of+HCN+channels.">Organisation of the mouse sinoatrial node: structure and expression of HCN channels.</a> Cardiovascular Research. 73 (4), 729-738 (2007).</li><li>Muller-Taubenberger, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+fluorescent+protein+tags+as+reporters+in+live-cell+imaging+studies.">Application of fluorescent protein tags as reporters in live-cell imaging studies.</a> Methods Mol Biol. 346, 229-246 (2006).</li><li>Müller-Taubenberger, A., Ishikawa-Ankerhold, H. C., Eichinger, L., Rivero, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dictyostelium discoideum Protocol. , 93-112 (2013).</li><li>Shaw, P. J., Pawley, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook Of Biological Confocal Microscopy. , 453-467 (2006).</li><li>Huff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Airyscan+detector+from+ZEISS:+confocal+imaging+with+improved+signal-to-noise+ratio+and+super-resolution.">The Airyscan detector from ZEISS: confocal imaging with improved signal-to-noise ratio and super-resolution.</a> Nature Methods. 12 (12), (2015).</li><li>Rysevaite, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+characterization+of+the+intrinsic+cardiac+neural+plexus+in+whole-mount+mouse+heart+preparations.">Immunohistochemical characterization of the intrinsic cardiac neural plexus in whole-mount mouse heart preparations.</a> Heart Rhythm. 8 (5), 731-738 (2011).</li><li>Acar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+imaging+of+bone+marrow+shows+non-dividing+stem+cells+are+mainly+perisinusoidal.">Deep imaging of bone marrow shows non-dividing stem cells are mainly perisinusoidal.</a> Nature. 526 (7571), 126-130 (2015).</li><li>Brahmajothi, M. V., Morales, M. J., Campbell, D. L., Steenbergen, C., Strauss, H. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+distribution+of+voltage-gated+ion+channels+in+ferret+sinoatrial+node.">Expression and distribution of voltage-gated ion channels in ferret sinoatrial node.</a> Physiological Genomics. 42 (2), 131-140 (2010).</li><li>Mesirca, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+arrhythmia+induced+by+genetic+silencing+of+'funny'+(f)+channels+is+rescued+by+GIRK4+inactivation.">Cardiac arrhythmia induced by genetic silencing of 'funny' (f) channels is rescued by GIRK4 inactivation.</a> Nature Communication. 5, 4664 (2014).</li><li>Liang, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HCN4+dynamically+marks+the+first+heart+field+and+conduction+system+precursors.">HCN4 dynamically marks the first heart field and conduction system precursors.</a> Circulation Research. 113 (4), 399-407 (2013).</li><li>Verheule, S., Kaese, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+diversity+in+the+heart:+insights+from+transgenic+mouse+models.">Connexin diversity in the heart: insights from transgenic mouse models.</a> Frontiers in Pharmacology. 4, 81 (2013).</li><li>van der Velden, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+gap+junctions+and+connexins:+their+role+in+atrial+fibrillation+and+potential+as+therapeutic+targets.">Cardiac gap junctions and connexins: their role in atrial fibrillation and potential as therapeutic targets.</a> Cardiovascular Research. 54 (2), 270-279 (2002).</li></ol>

Hjertets anatomi er traditionelt blevet undersøgt ved hjælp af tynde histologiske sektioner11. Disse metoder bevarer imidlertid ikke ledningssystemets tredimensionelle struktur og giver således kun 2D-information. Den helmonterede immunfluorescensfarvningsprotokol, der er beskrevet her, gør det muligt at overvinde disse begrænsninger og kan rutinemæssigt bruges til SAN- og AVN-billeddannelse.
I sammenligning med standardmetoder såsom konventionel immunhistokemi, ...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62058">Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse</a>. The Authors section was updated from:
Ruibing Xia1,2,3 
Julia Vlcek1,2 
Julia Bauer1,2,3 
Stefan K&#228;&#228;b1,3 
Hellen Ishikawa-Ankerhold1,2 
Dominic Adam van den Heuvel1,2 
Christian Schulz1,2,3 
Steffen Massberg1,2,3 
Sebastian Clauss1,2,3 
1University Hospital Munich, Department of Medicine I, Ludwig Maximilian University Munich 
2Walter Brendel Center of Experimental Medicine, Ludwig Maximilian University Munich 
3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance
to:
Ruibing Xia1,2,3 
Julia Vlcek1,2 
Julia Bauer1,2,3 
Stefan K&#228;&#228;b1,3 
Hellen Ishikawa-Ankerhold1,2 
Dominic Adam van den Heuvel1,2 
Christian Schulz1,2,3 
Steffen Massberg1,2,3 
Sebastian Clauss1,2,3 
1University Hospital Munich, Department of Medicine I, Ludwig Maximilians University (LMU) Munich 
2Institute of Surgical Research at the Walter Brendel Center of Experimental Medicine, University Hospital Munich, Ludwig Maximilians University (LMU) Munich 
3German Center for Cardiovascular Research (DZHK), Partner Site Munich, Munich Heart Alliance

An erratum was issued for:&#160;<a href="https://www.jove.com/t/62058">Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse</a>. The Authors section was updated.

Erratum: Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

Arytmier er almindelige sygdomme, der rammer millioner af mennesker, og er årsagen til betydelig sygelighed og dødelighed over hele verden. På trods af enorme fremskridt inden for behandling og forebyggelse, såsom udvikling af hjertepacemakere, er behandling af arytmier fortsat udfordrende, primært på grund af den meget begrænsede viden om underliggende sygdomsmekanismer 1,2,3. En bedre forståelse af både den normale elektrofysiologi og patofysiologien af arytmier kan bidrage til at udvikle nye, innovative og kausale behandlingsstrategier i fremtiden. For at omfattende studere arytmogenese er det desuden vigtigt at lokalisere og visualisere det specifikke hjerteledningssystem i dyremodeller som musen, da mus er meget udbredt i elektrofysiologiforskning.
De vigtigste dele af hjerteledningssystemet er sinoatriale knude (SAN), hvor den elektriske impuls genereres i specialiserede pacemakerceller, og den atrioventrikulære knude (AVN), som er den eneste elektriske forbindelse mellem atrierne og ventriklerne4. Når de elektrofysiologiske egenskaber af SAN og AVN ændres, kan arytmier såsom syg sinussyndrom eller atrioventrikulær blok forekomme, hvilket kan føre til hæmodynamisk forringelse, synkope og endda død og dermed understrege den væsentlige rolle af både SAN og AVN i elektrofysiologi og arytmogenese5.
Omfattende undersøgelser af SAN eller AVN kræver en præcis lokalisering og visualisering af begge strukturer, ideelt set inden for deres fysiologiske miljø. På grund af deres lille størrelse og placering inden for det arbejdende myokardium, uden at etablere en klar makroskopisk synlig struktur, er det imidlertid udfordrende at studere anatomi og elektrofysiologi af SAN og AVN. Anatomiske landemærker kan bruges til groft at identificere regionen, der indeholder SAN og AVN 6,7,8. Kort sagt er SAN placeret i inter-caval-regionen i højre atrium ved siden af den muskulære crista terminalis (CT), AVN er placeret inden for Koch-trekanten etableret af tricuspidventilen, ostium i koronar sinus og senen af Todaro. Indtil videre blev disse anatomiske landemærker hovedsageligt brugt til at lokalisere, fjerne og derefter studere SAN og AVN som individuelle strukturer (f.eks. Ved konventionel histologi). For bedre at forstå den komplekse elektrofysiologi af SAN og AVN (fx regulatoriske virkninger af tilstødende celler i det arbejdende myokardium) er det imidlertid nødvendigt at studere ledningssystemerne inden for det fysiologiske 3D-miljø.
Helmonteret immunfluorescensfarvning er en metode, der bruges til at studere anatomiske strukturer in situ , samtidig med at integriteten af det omgivende vævbevares 9. Ved at udnytte konfokal mikroskopi og billedanalysesoftware kan SAN og AVN visualiseres med fluorescerende mærkede antistoffer rettet mod ionkanaler, der specifikt udtrykkes i disse regioner.
Følgende protokol forklarer de nødvendige trin for at udføre en veletableret helmonteret farvningsmetode til lokalisering og visualisering af SAN- og AVN-mikroskoper. Specifikt beskriver denne protokol, hvordan (1) at lokalisere SAN og AVN ved anatomiske landemærker for at forberede disse prøver til farvning og mikroskopianalyse, (2) at udføre helmonteret immunfluorescensfarvning af referencemarkørerne HCN4 og Cx43 (3) for at forberede SAN- og AVN-prøver til konfokalmikroskopi (4) for at udføre konfokal billeddannelse af SAN og AVN. Vi beskriver også, hvordan denne protokol kan ændres til at omfatte yderligere farvning af omgivende arbejdende myokardium eller ikke-myocytceller, såsom autonome nervefibre, hvilket muliggør en grundig undersøgelse af hjerteledningssystemet i hjertet.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Anesthesia</td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer system&#160;</td>
			<td>Hugo Sachs Elektronik</td>
			<td>34-0458, 34-1030, 73-4911, 34-0415, 73-4910</td>
			<td>Includes an induction chamber, a gas evacuation unit and charcoal filters</td>
		</tr>
		<tr>
			<td>Modified Bain circuit</td>
			<td>Hugo Sachs Elektronik</td>
			<td>73-4860</td>
			<td>Includes an anesthesia mask for mice</td>
		</tr>
		<tr>
			<td>Surgical Platform</td>
			<td>Kent Scientific</td>
			<td>SURGI-M</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">In vivo instrumentation</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11295-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissors</td>
			<td>Fine Science Tools</td>
			<td>91500-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue forceps</td>
			<td>Fine Science Tools</td>
			<td>11051-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue pins</td>
			<td>Fine Science Tools</td>
			<td>26007-01</td>
			<td>Could use 27G needles as a substitute</td>
		</tr>
		<tr>
			<td colspan="4">General lab instruments</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>Sunlab</td>
			<td>D-8040</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer</td>
			<td>IKA&#160;</td>
			<td>RH basic</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette,volume 10 &#181;L, 100 &#181;L, 1000 &#181;L</td>
			<td>Eppendorf</td>
			<td>Z683884-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Microscopes</td>
		</tr>
		<tr>
			<td>Dissection stereo- zoom microscope&#160;</td>
			<td>VWR</td>
			<td>10836-004</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Scanning Confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM 800</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Software</td>
		</tr>
		<tr>
			<td>Imaris 8.4.2</td>
			<td>Oxford instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ZEN 2.3 SP1 black</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">General Lab Material</td>
		</tr>
		<tr>
			<td>0.2 &#181;m syringe filter</td>
			<td>Sartorius</td>
			<td>17597</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>27G needle</td>
			<td>BD Microlance 3</td>
			<td>300635</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Polypropylene conical Tube</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>5ml Syringe</td>
			<td>Braun</td>
			<td>4606108V</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slips</td>
			<td>Thermo Scientific</td>
			<td>7632160</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Tubes</td>
			<td>Eppendorf</td>
			<td>30121872</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Chemicals</td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>Sigma</td>
			<td>20-158</td>
			<td>Components of TEA</td>
		</tr>
		<tr>
			<td>16% Formaldehyde Solution</td>
			<td>Thermo Scientific&#160;</td>
			<td>28908</td>
			<td>use as a 4% solution&#160;</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>Merck</td>
			<td>100063</td>
			<td>Components of TEA</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Biozym</td>
			<td>850070</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (1X) Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Gibco</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Sigma</td>
			<td>NS02L</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma</td>
			<td>S1888-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-base</td>
			<td>Roche</td>
			<td>TRIS-RO</td>
			<td>Components of TEA</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>T8787-250ml</td>
			<td>Diluted to 1% in PBS</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Drugs</td>
		</tr>
		<tr>
			<td>Fentanyl 0.5&#160;mg/10&#160;mL</td>
			<td>Braun Melsungen</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane 1 mL/mL</td>
			<td>Cp-pharma</td>
			<td>31303</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen 5 L</td>
			<td>Linde</td>
			<td>2020175</td>
			<td>Includes a pressure regulator</td>
		</tr>
		<tr>
			<td colspan="4">Antibodies</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG Alexa Fluor&#160;488&#160;</td>
			<td>Cell Signaling Technology</td>
			<td>#4412</td>
			<td>diluted to 1:200</td>
		</tr>
		<tr>
			<td>Goat anti-Rat IgG Alexa Fluor 647</td>
			<td>Invitrogen</td>
			<td>#A-21247</td>
			<td>diluted to 1:200</td>
		</tr>
		<tr>
			<td>Hoechst 33342, Trihydrochloride, Trihydrate (DAPI)</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td>diluted to 1:1000</td>
		</tr>
		<tr>
			<td>Rabbit Anti-Connexin-43</td>
			<td>Sigma</td>
			<td>C6219</td>
			<td>diluted to 1:200</td>
		</tr>
		<tr>
			<td>Rat anti-HCN4 (SHG 1E5)</td>
			<td>Invitrogen</td>
			<td>MA3-903</td>
			<td>diluted to 1:200</td>
		</tr>
		<tr>
			<td colspan="4">Other</td>
		</tr>
		<tr>
			<td>Plastic ring</td>
			<td></td>
			<td></td>
			<td>Self-designed and 3D printed</td>
		</tr>
		<tr>
			<td>Plasticine</td>
			<td>Cernit</td>
			<td>49655005</td>
			<td></td>
		</tr>
		<tr>
			<td>Silikonpasten, Baysilone</td>
			<td>VWR</td>
			<td>291-1220</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Animals</td>
		</tr>
		<tr>
			<td>Mouse, C57BL/6</td>
			<td>The Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

whole-mount immunofluorescence staining, confocal imaging and 3d reconstruction of the sinoatrial and atrioventricular node in the mouse

Det elektriske signal, der fysiologisk genereres af pacemakerceller i sinoatriale knude (SAN), ledes gennem ledningssystemet, som omfatter den atrioventrikulære knude (AVN), for at tillade excitation og sammentrækning af hele hjertet. Enhver dysfunktion af enten SAN eller AVN resulterer i arytmier, hvilket indikerer deres grundlæggende rolle i elektrofysiologi og arytmogenese. Musemodeller anvendes i vid udstrækning i arytmiforskning, men den specifikke undersøgelse af SAN og AVN er fortsat udfordrende.
SAN er placeret ved krydset mellem crista terminalis og den overlegne vena cava, og AVN er placeret ved toppen af Koch-trekanten, dannet af åbningen af koronar sinus, tricuspid annulus og senen af Todaro. På grund af den lille størrelse forbliver visualisering ved konventionel histologi imidlertid udfordrende, og det tillader ikke undersøgelse af SAN og AVN inden for deres 3D-miljø.
Her beskriver vi en helmonteret immunfluorescenstilgang, der muliggør lokal visualisering af mærket mus SAN og AVN. Helmonteret immunfluorescensfarvning er beregnet til mindre sektioner af væv uden behov for manuel sektionering. Til dette formål dissekeres musehjertet, med uønsket væv fjernet, efterfulgt af fiksering, permeabilisering og blokering. Celler i ledningssystemet i SAN og AVN farves derefter med et anti-HCN4-antistof. Konfokal laserscanningsmikroskopi og billedbehandling tillader differentiering mellem nodalceller og fungerende kardiomyocytter og klart lokaliserer SAN og AVN. Desuden kan yderligere antistoffer kombineres for også at mærke andre celletyper, såsom nervefibre.
Sammenlignet med konventionel immunohistologi bevarer helmonteret immunofluorescensfarvning den anatomiske integritet af hjerteledningssystemet, hvilket muliggør undersøgelse af AVN; Især så i deres anatomi og interaktioner med de omgivende arbejdende myokardium og ikke-myocytceller.

Ved at bruge protokollen skitseret ovenfor kan konfokal mikroskopibilleddannelse af både SAN og AVN udføres pålideligt. Specifik farvning af ledningssystemet ved hjælp af fluorescerende antistoffer rettet mod HCN4 og farvning af det arbejdende myokardium ved hjælp af fluorescerende antistoffer rettet mod Cx43 muliggør klar identifikation af SAN (figur 5, video 1) og AVN (figur 6, video 2) inden for den inta...

Watch this Scientific Journal Video about Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse at JoVE.com

Whole-Mount Immunofluorescence Staining, Confocal Imaging and 3D Reconstruction of the Sinoatrial and Atrioventricular Node in the Mouse

Vi leverer en trin-for-trin protokol til helmonteret immunfluorescensfarvning af sinoatriale knude (SAN) og atrioventrikulær knude (AVN) i murine hjerter.

Helmonteret immunfluorescensfarvning, konfokal billeddannelse og 3D-rekonstruktion af den sinoatriale og atrioventrikulære knude i musen

The electrical signal physiologically generated by pacemaker cells in the sinoatrial node (SAN) is conducted through the conduction system, which includes ...

Research

JoVE Journal

Medicine

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Technical Aspects of the Mouse Aortocaval Fistula

Tekniske aspekter af Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Cell Death Associated with Abnormal Mitosis Observed by Confocal Imaging in Live Cancer Cells

Celledød forbundet med abnorme Mitosis Observeret ved konfokal Imaging i Live kræftceller

Phenanthrene derivatives acting as potent PARP1 inhibitors prevented the bi-focal clustering of supernumerary centrosomes in multi-centrosomal human ...

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Diagnostisk Ultralydbilleddannelse of Mouse Membran Funktion

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo: A Model of Single Vessel Stroke

Rose Bengal Photothrombosis by Confocal Optical Imaging In Vivo:  A Model of Single Vessel Stroke

Rose Bengal Photothrombosis ved Konfokal Optical Imaging<em&gt; In vivo</em&gt;: En model af Single Vessel Stroke

In vivo imaging techniques have increased in utilization due to recent advances in imaging dyes and optical technologies, allowing for the ability to ...

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

Imaging- og flowcytometri-baserede Analyse af Cell holdning og Cell Cycle i 3D Melanom Sfæroider

Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to ...

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaucoma-inducing Procedure in an In Vivo Rat Model and Whole-mount Retina Preparation

Glaukom-inducerende Procedure i en<em&gt; In vivo</em&gt; Rat Model og Whole-mount Retina Forberedelse

Glaucoma is a disease of the central nervous system affecting retinal ganglion cells (RGCs). RGC axons making up the optic nerve carry visual input to the ...

A Postoperative Evaluation Guideline for Computer-Assisted Reconstruction of the Mandible

En postoperativ evalueringsretningslinje for computerstøttet rekonstruktion af mandible

Valid comparisons of postoperative accuracy results in computer-assisted reconstruction of the mandible are difficult due to heterogeneity in imaging ...

Blood Circuit Reconstruction in an Abdominal Mouse Heart Transplantation Model

Blodkredsløb Genopbygning i en Abdominal Mouse Heart Transplantation Model

The surgical technique of heterotopic abdominal heart transplantation in mice is a standard model for research in transplantation immunology. Here, the ...

Refined CLARITY-Based Tissue Clearing for Three-Dimensional Fibroblast Organization in Healthy and Injured Mouse Hearts

Raffineret KLARHED-baseret væv Clearing for tre-dimensionelle Fibroblast Organisation i sunde og sårede Mouse Hearts

Cardiovascular disease is the most prevalent cause of mortality worldwide and is often marked by heightened cardiac fibrosis that can lead to increased ...