German Research Foundation (FOR2240 "(Lymph) Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye" to CC and LMH; HE 6743/2-1 and HE 7643/3-1 to LMH; CU47/6-1 to CC), German Cancer Aid (to LMH and CC), GEROK program University of Cologne (to SLS and LMH), and EU COST BM1302 "Joining Forces to Corneal Regeneration" (to CC).

L&#39;uso di tessuti umani deve essere esaminato e approvato da un comitato istituzionale di revisione o equivalente. Il lavoro qui descritto è stato approvato dal comitato etico locale e ha avuto l&#39;approvazione per l&#39;esame scientifico. Questo lavoro è stato eseguito in base alla Dichiarazione di Helsinki. I campioni sclerali umani sono stati ottenuti dagli occhi dei donatori globo (massimo post mortem 24 ore) presso la Banca degli Occhi del Dipartimento di Oftalmologia, Università di Colonia, in Germania. 1. Preparazione sperimentale <ol><li> Preparare 96% etanolo e tampone fosfato isotonico (PBS) in tubi diversi. Preparare gli anticorpi primari nella diluizione raccomandata di PBS contenente 2% di albumina sierica bovina (BSA) e mantenere tutte le soluzioni su ghiaccio. Preparare 100 microlitri per campione. </li><li> Pulire tutti gli strumenti e utilizzare bisturi taglienti. Dopo ripetute taglio con la stessa bisturi, cambiare il bisturi. </li><li> Ottenere 1-2 pinze Colibri, forbici dissezione micro scala, un # 10 bisturio oftalmica bisturi micro piuma, e quattro aghi 26 G. </li><li> Avvolgere un foglio di alluminio intorno a un piatto di polistirolo o utilizzare una piastra di sughero di apporre il tessuto. Lavorare sotto un stereomicroscopio binoculare. Lavorare sterile sotto un flusso laminare, se necessario. </li></ol> 2. Preparare la sclera <ol><li> Premere delicatamente la lampadina ed eseguire una trapanazione perforazione da parte corneosclerale, quindi ruotare il trapano. Ruotare il trapano con cura e in modo uniforme sulla superficie di eseguire un taglio altrettanto rotondo. Utilizzare un trapano di dimensioni 15,5 millimetri. Tagliare i restanti allegati con forbici curve. Rimuovere la trapanazione corneosclerale. Questo si tradurrà in una lampadina anteriormente aperto. <ol><li> Mettere la sclera su un tampone con la parte aperta verso l&#39;alto. Rimuovere la retina e uvea, con pinze Colibri di tirare fuori entrambi gli strati (retina e uvea) dalla sclera. Non lasciare uvea pigmentato sulla sclera interiore. Utilizzare forbici curve per rimuovere la retina e uvea dalla papilla. Rimuovere il remaining congiuntiva, muscoli estrinseci, e la capsula di tenone dalla sclera superficiale. </li></ol></li><li> Rimuovere il campione sclerale nella dimensione desiderata utilizzando le forbici straight-tipo e le pinze Colibri. Prendere circa 2 cm 2 campioni sclerali dimensioni da luoghi diversi. A causa delle piccole dimensioni, tenere la sclera delicatamente e tagliare fuori la dimensione richiesta come quadrati utilizzando le forbici straight-tipo. La trapanazione corneosclerale definisce il margine anteriore dei campioni sclerali. <ol><li> Evitare afferrando ripetuto con le pinze come l&#39;area in cui il tessuto è stato afferrato non è adatto per le analisi microscopia confocale. Preparare il numero di campioni in funzione del tipo di esperimento (es anteriormente vs. posteriormente, confrontare Figura 1) e la quantità di anticorpi utilizzati. </li></ol></li><li> Mettere i campioni in provette da 1,5 ml per un uso successivo. Fissare i campioni in 1,5 ml di etanolo 96% per 15 minuti, e poi lavarli tre volte ciascuno per 5 minin 1,5 ml di PBS sul shaker. Se si lavora con il tessuto congelato, eseguire questo passaggio prima di scatto di congelamento. </li><li> Utilizzare tessuto fresco. Tuttavia, se ciò non è possibile, a scatto congelare la sclera in azoto liquido e la tiene a -20 ° C per un uso successivo. Se si lavora con il tessuto congelato, scongelare prima dell&#39;uso. Evitare ripetuti cicli di congelamento scongelamento e. </li><li> Mantenere ogni campione in PBS contenente 1,5 ml 5% BSA a temperatura ambiente per 2 ore. Questo passo induce un rigonfiamento del campione che è utile per la laminazione e impedisce anche legame aspecifico. </li><li> Dopo che i campioni hanno gonfiato, preparare le piazze sclerali per la laminazione. Lavorare sotto un microscopio, ad esempio uno stereo microscopio binoculare. Fissare i campioni sclerali posteriori alla membrana in polistirolo avvolto in un foglio di alluminio o la piastra di sughero con gli aghi 26 g. </li><li> Piegare i bordi degli aghi in modo da non interferire con il campo visivo sotto il microscopio. </li><li> Laminare il tutto spessore SAMP scleraleles. <ol><li> In primo luogo, tenere il bordo sclerale anteriore con pinze Colibri. Poi accuratamente tagliato un sottile strato di sclera superficiale utilizzando il bisturi # 10, il oftalmica bisturi micro piuma, o una lama spatola. Tenere il bisturi orizzontale e tagliare lo strato superficiale più sottile possibile dallo strato sottostante. </li><li> Sezionare uno strato sottile sclerale dalla piazza sclerale. Questo metodo è simile alle tecniche chirurgiche standard per preparare un lembo durante trabeculectomia e dovrebbe tradursi in strati di spessore 30-80 micron (confronta figura 2). </li></ol></li><li> Impedire essiccamento della sclera aggiungendo piccole quantità di PBS al tessuto, per esempio 50 microlitri di PBS utilizzando una pipetta. </li><li> Mettere i livelli di taglio in 100 pl di PBS nella piastra da 96 pozzetti e l&#39;etichetta sia l&#39;orientamento (verso extern e intern) e lo strato (superficiale e profonda) ad esempio con un colore impermeabile. </li><li> Ripetere i passaggi 2,7-2,9 fino a quando il tessuto è completamente laminato. &lt;/ Li&gt; </ol> 3. Eseguire immunoistochimica <ol><li> Aggiungere 100 ml di anticorpi primari necessari alla diluizione raccomandata. Qui, per esempio CD utilizzo 31 (topo monoclonale anti umano) e anticorpi LYVE1 (coniglio anti umano), sia in una diluizione di 1: 100 in PBS (contenente 2% BSA) e incubare a 4 ° C durante la notte. Per cambiare il supporto nella piastra da 96 pozzetti, tenere la pipetta alla parete del pozzo e rimuovere il liquido. </li><li> Il giorno dopo, lavare i campioni per tre volte ciascuno per 5 min con 200-300 ml di PBS sul shaker. Aggiungere 100 pl di corrispondenti anticorpi secondari; qui utilizzare capra anti topo fluoresceina isotiocianato (FITC) e di capra anti coniglio cianina coloranti 3 (Cy3), e incubare i campioni a temperatura ambiente per 1-2 ore. Diluire gli anticorpi secondari 1: 300 in PBS contenente 2% di siero di capra. </li><li> Lavare i campioni di nuovo tre volte ciascuno per 5 min in 200-300 ml di PBS sul shaker. </li><li> Eseguire colorazione nucleo, ad esempio, </em&gt;. 4 &#39;, 6-diamidino-2-fenilindolo (DAPI) (diluizione 1: 2.000, 100 microlitri in ciascun pozzetto ed incubare 10 min a temperatura ambiente), poi lavare ancora 2 volte in PBS 200-300 microlitri l&#39;agitatore. </li><li> Trasferire i campioni su vetrini da microscopio, incorporare loro in 1-2 gocce di mezzo di montaggio fluorescente, aggiungere un vetrino, sigillarla coprendo i bordi con vernice trasparente, e conservare a 4 ° C per un uso successivo o esaminare direttamente i vetrini con il confocale microscopio. </li><li> Utilizzare un microscopio confocale (o equivalente) per analizzare i campioni. Utilizzare l&#39;ingrandimento desiderato, ad esempio 10-40X ingrandimento. </li><li> Per verificare lo spessore dei campioni, misurare con microscopia confocale utilizzando Z-Stack. La quantità di possibili sezioni sclerali dipende dalla posizione della sclera, come lo spessore diverso equatoriale e posteriormente (confrontare introduzione). </li></ol>

<ol>
	<li>Schlereth, S. L., 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=Enrichment+of+lymphatic+vessel+endothelial+hyaluronan+receptor+1+(LYVE1)-positive+macrophages+around+blood+vessels+in+the+normal+human+sclera.">Enrichment of lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1)-positive macrophages around blood vessels in the normal human sclera.</a> Invest Ophthalmol Vis Sci. 55 (2), 865-872 (2014).</li><li>Schlereth, S. L., 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=Absence+of+lymphatic+vessels+in+the+developing+human+sclera.">Absence of lymphatic vessels in the developing human sclera.</a> Exp Eye Res. 125, 203-209 (2014).</li><li>Hos, D., Cursiefen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lymphatic+vessels+in+the+development+of+tissue+and+organ+rejection.">Lymphatic vessels in the development of tissue and organ rejection.</a> Adv Anat Embryol Cell Biol. 214, 119-141 (2014).</li><li>Hos, D., Schlereth, S. L., Bock, F., Heindl, L. M., Cursiefen, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antilymphangiogenic+therapy+to+promote+transplant+survival+and+to+reduce+cancer+metastasis:+what+can+we+learn+from+the+eye.">Antilymphangiogenic therapy to promote transplant survival and to reduce cancer metastasis: what can we learn from the eye.</a> Semin Cell Dev Biol. , (2014).</li><li>Streilein, 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=Immune+privilege+as+the+result+of+local+tissue+barriers+and+immunosuppressive+microenvironments.">Immune privilege as the result of local tissue barriers and immunosuppressive microenvironments.</a> Curr Opin Immunol. 5 (3), 428-432 (1993).</li><li>Streilein, J. W., Niederkorn, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+anterior+chamber-associated+immune+deviation+requires+an+intact,+functional+spleen.">Induction of anterior chamber-associated immune deviation requires an intact, functional spleen.</a> J Exp Med. 153 (5), 1058-1067 (1981).</li><li>Streilein, J. W., Yamada, J., Dana, M. R., Ksander, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+chamber-associated+immune+deviation,+ocular+immune+privilege,+and+orthotopic+corneal+allografts.">Anterior chamber-associated immune deviation, ocular immune privilege, and orthotopic corneal allografts.</a> Transplant Proc. 31 (3), 1472-1475 (1999).</li><li>Keeley, F. W., Morin, J. D., Vesely, S. <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+collagen+from+normal+human+sclera.">Characterization of collagen from normal human sclera.</a> Exp Eye Res. 39 (5), 533-542 (1984).</li><li>Lee, R. E., Davison, P. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collagen+composition+and+turnover+in+ocular+tissues+of+the+rabbit.">Collagen composition and turnover in ocular tissues of the rabbit.</a> Exp Eye Res. 32 (6), 737-745 (1981).</li><li>Moses, R. A., Grodzki, W. J., Starcher, B. C., Galione, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin+content+of+the+scleral+spur,+trabecular+mesh,+and+sclera.">Elastin content of the scleral spur, trabecular mesh, and sclera.</a> Invest Ophthalmol Vis Sci. 17 (8), 817-818 (1978).</li><li>Foster, C. S., Sainz de la Maza, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The sclera. , (2012).</li><li>Kiss, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Der+Blutkreislauf+des+Auges.">Der Blutkreislauf des Auges.</a> Ophthalmologica. 106, 225 (1943).</li><li>Ashton, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+study+of+Schlemm's+canal+and+aqueous+veins+by+means+of+neoprene+casts.+Part+I.+Aqueous+veins.">Anatomical study of Schlemm's canal and aqueous veins by means of neoprene casts. Part I. Aqueous veins.</a> Br J Ophthalmol. 35 (5), 291-303 (1951).</li><li>Ashton, N., Smith, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+study+of+Schlemm's+canal+and+aqueous+veins+by+means+of+neoprene+casts.+III.+Arterial+relations+of+Schlemm's+canal.">Anatomical study of Schlemm's canal and aqueous veins by means of neoprene casts. III. Arterial relations of Schlemm's canal.</a> Br J Ophthalmol. 37 (10), 577-586 (1953).</li><li>Ashton, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+study+of+Schlemm's+canal+and+aqueous+veins+by+means+of+neoprene+casts+II.+Aqueous+veins.">Anatomical study of Schlemm's canal and aqueous veins by means of neoprene casts II. Aqueous veins.</a> Br J Ophthalmol. 36 (5), 265-267 (1952).</li><li>Hayreh, S. S., Scott, W. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+iris+angiography.+II.+Disturbances+in+iris+circulation+following+strabismus+operation+on+the+various+recti.">Fluorescein iris angiography. II. Disturbances in iris circulation following strabismus operation on the various recti.</a> Arch Ophthalmol. 96 (8), 1390-1400 (1978).</li><li>Virdi, P. S., Hayreh, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anterior+segment+ischemia+after+recession+of+various+recti.+An+experimental+study.">Anterior segment ischemia after recession of various recti. An experimental study.</a> Ophthalmology. 94 (10), 1258-1271 (1987).</li><li>Bron, A. J., Easty, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+angiography+of+the+globe+and+anterior+segment.">Fluorescein angiography of the globe and anterior segment.</a> Trans Ophthalmol Soc U K. 90, 339-367 (1970).</li><li>Ikegami, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescein+angiography+of+the+anterior+ocular+segment.+Part+1.+Hemodynamics+in+the+anterior+ciliary+vessels+(author's+transl).">Fluorescein angiography of the anterior ocular segment. Part 1. Hemodynamics in the anterior ciliary vessels (author's transl).</a> Nihon Ganka Gakkai Zasshi. 78 (7), 371-385 (1974).</li><li>Banerji, 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=LYVE-1,+a+new+homologue+of+the+CD44+glycoprotein,+is+a+lymph-specific+receptor+for+hyaluronan.">LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.</a> J Cell Biol. 144 (4), 789-801 (1999).</li><li>Breiteneder-Geleff, 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=Angiosarcomas+express+mixed+endothelial+phenotypes+of+blood+and+lymphatic+capillaries:+podoplanin+as+a+specific+marker+for+lymphatic+endothelium.">Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.</a> Am J Pathol. 154 (2), 385-394 (1999).</li></ol>

The authors declare no financial disclosures. The contents of this article are solely the responsibility of the authors.

Laminazione la sclera umana è un metodo per eseguire la microscopia confocale in questo tessuto. Un punto critico in questo processo è l&#39;uso di etanolo invece di formalina per l&#39;apposizione del tessuto. Nella nostra esperienza, i risultati migliori si ottengono quando si utilizza etanolo invece di formalina per fissare. bisturi Blunt aggravano la procedura e deve essere evitato. Analogamente, il prosciugamento della sclera dovrebbe essere evitata, in quanto complica la procedura e riduce la qualità delle imma...

La sclera è lo strato esterno rigido che copre l&#39;occhio, che è fatto di tessuto connettivo denso. Aiuta a proteggere le strutture intraoculari e di mantenere la pressione intraoculare. Così, la sclera è essenziale per una visione chiara. E &#39;privo di vasi linfatici 1,2 e, quindi, forma un bordo esterno libero-linfatica tra essa e l&#39;occhio interiore senza linfatica 3-7. Esso fornisce inoltre i siti di attacco per i muscoli estrinseci, condividendo in tal modo somiglianze anatomiche con tendini. Poiché la sclera consiste principalmente di densi fasci di collagene di tipo I e ha un numero inferiore di collagene di tipo III, IV, V, VI, VIII 8,9 ed elastina 10,11, questo tessuto non è facile da usare per immunoistochimica. Anatomicamente, la sclera può essere suddiviso in tre strati principali: (1) il superficiale episclera vascolarizzato, trovati sotto la congiuntiva e la capsula di tenone e verso i lati e the posteriore dell&#39;occhio rivolto verso l&#39;orbita; (2) stroma sclerale, la parte principale della sclera; e (3) il fusca lamina, che è un sottile strato pigmentato situato direttamente sopra la uvea. La nostra conoscenza anatomica sulla sclera deriva principalmente dalla prima metà del 20 ° secolo. A quel tempo, i ricercatori hanno studiato l&#39;anatomia del sistema vascolare prevalentemente utilizzando iniezioni di inchiostro India 12 e vascolare fusione 13-15. Successivamente, è stato studiato in studi angiografici 16-19. Da quel momento, le tecniche più vecchie sono state migliorate e nuove sono state sviluppate che ci hanno permesso di completare una precedente conoscenza anatomica. Ad esempio, è stato solo di circa un decennio da quando abbiamo avuto tali marcatori linfatici affidabili come linfatico vascolare acido ialuronico specifico recettore-1 (LYVE1) 20 o 21 podoplanin. La microscopia confocale offre nuove possibilità per lo studio delle caratteristiche anatomiche dei diversi tiUESTIONI dell&#39;occhio. Esso consente più macchie da utilizzare per differenziare marcatori di cellule o per la localizzazione delle cellule in relazione ai vasi sanguigni e altre strutture anatomiche. Esso fornisce una panoramica quando il campione è di dimensioni maggiori e ci permette di scandiamo un campione quando alla ricerca di un tipo specifico di cellula. Con la tecnologia Z-Stack, microscopia confocale può essere utilizzato per i campioni fino a 100-200 micron. La sclera differisce di spessore tra 0,3 mm dietro le inserzioni muscolari e 1 mm al polo posteriore 11. A causa sia suo spessore e opacità, la sclera non è adatto per la microscopia confocale con metodi tradizionali. Per rimediare a questo, i tessuti sclerali sono stati stratificati per consentire la loro analisi con microscopia confocale. Questa tecnologia è utile per una migliore comprensione di entrambe le situazioni fisiologiche e patologiche nella sclera umana.

<table><tbody><tr><td>96% ethanol</td><td>Merck Chemicals, Darmstadt, Germany</td><td>P075.4</td><td></td></tr><tr><td>binocular stereo microscope&amp;nbsp;</td><td>Motic, Hongkong, China</td><td>n.a</td><td></td></tr><tr><td>26 G needles&amp;nbsp;</td><td>Terumo, Leuven, Belgium</td><td>303800</td><td></td></tr><tr><td>15.5 mm trepan</td><td>Geuder, Heidelberg, Germany</td><td>n.a</td><td></td></tr><tr><td>no.10 scalpel&amp;nbsp;</td><td>Feather, pfm medical, Osaka, Japan</td><td>2E+08</td><td></td></tr><tr><td>ophthalmic scalpel micro feather&amp;nbsp;</td><td>Feather, pfm medical, Osaka, Japan</td><td>no. 7657BR</td><td></td></tr><tr><td>CD 31 antibody (monoclonal mouse anti human)</td><td>Dako, USA</td><td>IR610</td><td></td></tr><tr><td>LYVE1 antibody&amp;nbsp; (polyclonal rabbit anti human)</td><td>Zytomed, Germany</td><td>RBK014-05</td><td></td></tr><tr><td>goat anti mouse FITC antibody</td><td>Sigma Aldrich, Steinheim, Germany</td><td>F0257</td><td></td></tr><tr><td>goat anti rabbit Cy3 antibody</td><td>Dianova, Germany</td><td>111-165-003</td><td></td></tr><tr><td>Goat Serum normal</td><td>Dako, Glostrup, Denmark</td><td>X090710-8</td><td></td></tr><tr><td>DAPI</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>6335.1</td><td></td></tr><tr><td>microscope slides&amp;nbsp;</td><td>Engelbrecht, Ederm&amp;uuml;nde, Germany</td><td>WC7695002</td><td></td></tr><tr><td>Coverslips 24 mm x 24 mm</td><td>Th. Gayer, Lohmar, Germany</td><td>7695026</td><td></td></tr><tr><td>DAKO fluorescent mounting medium&amp;nbsp;</td><td>DAKO, USA</td><td>S3023</td><td></td></tr><tr><td>LSM Meta 510 confocal microscopy&amp;nbsp;</td><td>Carl Zeiss AG, Jena, Germany</td><td>n.a</td><td></td></tr></tbody></table>

using a laminating technique to perform confocal microscopy of the human sclera

La sclera è un tessuto connettivo denso che copre e protegge l&#39;occhio. Essa consiste essenzialmente di fasci di collagene denso (tipo I, III, IV, V, VI, e VII). Grazie alla sua autofluorescenza, opacità e spessore, non è stato trovato adatto per la microscopia confocale. Un approccio alternativo a quello qui presentato, che utilizza sclera fissati in formalina inclusi in paraffina per immunoistochimica, ha problemi tecnici, in particolare per preriscaldare il tessuto per il recupero antigene. Dal momento che la sclera è relativamente povero di entrambe le cellule e dei vasi, l&#39;uso di campioni di tessuto più grandi è stato esplorato per aiutare a prevenire le cellule che si affacciano e per capire la loro localizzazione in relazione alle navi e altri siti anatomici. Per consentire l&#39;analisi di campioni di tessuto grandi al microscopio confocale, una tecnica di laminazione è stata eseguita per creare strati sottili dalla sclera. Dopo l&#39;analisi dei risultati dei vasi sanguigni CD31 e vasi linfatici endoteliale hyaluRonan recettore 1 (LYVE1) cellule positive, per le quali è stato ottenuto l&#39;approvazione per l&#39;esame scientifico, i vantaggi ei limiti di questo metodo sono discussi.

Negli esperimenti rappresentativi condotti qui, ci sono benefici dimostrabili derivanti dall&#39;uso di questa particolare tecnica di laminazione. Il primo esperimento illustra la rete diversificata del plesso vaso sanguigno episclerale in tre immagini rappresentative (Figura 3). I vasi sono positivi per CD31. Il secondo esperimento mostra cellule immunitarie, in particolare le cellule LYVE1 + del episclera e ...

Watch this Scientific Journal Video about Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera at JoVE.com

Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.

Utilizzando una tecnica di laminazione per l&#39;esecuzione di Microscopia confocale della sclera umana


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

using-laminating-technique-to-perform-confocal-microscopy-human

Research

JoVE Journal

Medicine

7.5K Views.  University of Cologne. Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used. 

Video: Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Corneal Confocal Microscopy: A Novel Non-invasive Technique to Quantify Small Fibre Pathology in Peripheral Neuropathies

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. Conventional methods assess neurological deficits and electrophysiology and quantitative sensory testing quantifies functional alterations to detect neuropathy. However, the earliest damage appears to be to the small fibres and yet these tests primarily assess large fibre dysfunction and have a limited ability to demonstrate regeneration and repair. The only techniques which allow a direct examination of unmyelinated nerve fibre damage and repair are sural nerve biopsy with electron microscopy and skin-punch biopsy. However, both are invasive procedures and require lengthy laboratory procedures and considerable expertise. Corneal Confocal microscopy is a non-invasive clinical technique which provides in-vivo imaging of corneal nerve fibres. We have demonstrated early nerve damage, which precedes loss of intraepidermal nerve fibres in skin biopsies together with stratification of neuropathic severity and repair following pancreas transplantation in diabetic patients. We have also demonstrated nerve damage in idiopathic small fibre neuropathy and Fabry's disease.

Corneal Confocal microscopy is a non-invasive clinical technique which may be used to quantify C fibre damage to diagnose and stratify patients with increasing neuropathic severity.

Microscopia corneale confocale: A Novel tecnica non invasiva per quantificare Piccole Fibre Patologia neuropatie periferiche

The accurate quantification of peripheral neuropathy is important to define at risk patients, anticipate deterioration, and assess new therapies. ...

Ricerca

Medicina

Three-dimensional Rendering and Analysis of Immunolabeled, Clarified Human Placental Villous Vascular Networks

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that makes up much of the parenchyma of the human placenta. The distal villous capillary network is the terminus of the fetal blood supply after several generations of branching of vessels extending out from the umbilical cord. This network has a contiguous cellular sheath, the syncytial trophoblast barrier layer, which prevents mixing of fetal blood and the maternal blood in which it is continuously bathed. Insults to the integrity of the placental capillary network, occurring in disorders such as maternal diabetes, hypertension and obesity, have consequences that present serious health risks for the fetus, infant, and adult. To better define the structural effects of these insults, a protocol was developed for this study that captures capillary network structure on the order of 1 - 2 mm3 wherein one can investigate its topological features in its full complexity. To accomplish this, clusters of terminal villi from placenta are dissected, and the trophoblast layer and the capillary endothelia are immunolabeled. These samples are then clarified with a new tissue clearing process which makes it possible to acquire confocal image stacks to z- depths of ~1 mm. The three-dimensional renderings of these stacks are then processed and analyzed to generate basic capillary network measures such as volume, number of capillary branches, and capillary branch end points, as validation of the suitability of this approach for capillary network characterization.

This study presents a protocol for the reversible tissue clearing, immunostaining, 3D-rendering and analysis of vascular networks in human placenta villi samples on the order of 1 - 2 mm3.

Rendering tridimensionale e l'analisi di Immunolabeled, chiarito reti vascolari Villous placentare umani

Nutrient and gas exchange between mother and fetus occurs at the interface of the maternal intervillous blood and the vast villous capillary network that ...

Biologia dello sviluppo

Light-sheet Microscopy for Three-dimensional Visualization of Human Immune Cells

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or tissues. Up to date, most in vitro systems rely on two-dimensional (2D) surfaces, such as cell-culture plates or coverslips. To optimally mimic physiological conditions in vitro, we utilize a simple 3D collagen matrix. Collagen is one of the major components of extracellular matrix (ECM) and has been widely used to constitute 3D matrices. For 3D imaging, the recently developed light-sheet microscopy technology (also referred to as single plane illumination microscopy) is featured with high acquisition speed, large penetration depth, low bleaching, and photocytotoxicity. Furthermore, light-sheet microscopy is particularly advantageous for long-term measurement. Here we describe an optimized protocol how to set up and handle human immune cells, e.g. primary human cytotoxic T lymphocytes (CTL) and natural killer (NK) cells in the 3D collagen matrix for usage with the light-sheet microscopy for live cell imaging and fixed samples. The procedure for image acquisition and analysis of cell migration are presented. A particular focus is given to highlight critical steps and factors for sample preparation and data analysis. This protocol can be employed for other types of suspension cells in a 3D collagen matrix and is not limited to immune cells.

Here, we present a protocol to visualize immune cells embedded in a three-dimensional (3D) collagen matrix using light-sheet microscopy. This protocol also elaborates how to track cell migration in 3D. This protocol can be employed for other types of suspension cells in the 3D matrix.

Microscopia di luce-foglio per visualizzazione tridimensionale delle cellule immuni umane

In vivo, activation, proliferation, and function of immune cells all occur in a three-dimensional (3D) environment, for instance in lymph nodes or ...

Immunologia e infezioni

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy (Conpokal) on Live Cells

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning and transmission electron microscope, to the invention of atomic force microscopy, and advances in fluorescent imaging, there have been substantial gains in technologies that enable the study of small materials. Conpokal is a portmanteau that combines confocal microscopy with atomic force microscopy (AFM), where a probe &#8220;pokes&#8221; the surface. Although each technique is extremely effective for the qualitative and/or quantitative image collection on their own, Conpokal provides the capability to test with blended fluorescence imaging and mechanical characterization. Designed for near simultaneous confocal imaging and atomic force probing, Conpokal facilitates experimentation on live microbiological samples. The added insight from paired instrumentation provides co-localization of measured mechanical properties (e.g., elastic modulus, adhesion, surface roughness) by AFM with subcellular components or activity observable through confocal microscopy. This work provides a step by step protocol for the operation of laser scanning confocal and atomic force microscopy, simultaneously, to achieve same cell, same region, confocal imaging, and mechanical characterization.

Near Simultaneous Laser Scanning Confocal and Atomic Force Microscopy Conpokal on Live Cells

Presented here is the protocol of the Conpokal technique, which combines confocal and atomic force microscopy into a single instrument platform. Conpokal provides same cell, same region, near simultaneous confocal imaging and mechanical characterization of live biological samples.

Microscopia confocale e a forza atomica (Conpokal) quasi simultanea su cellule vive

Techniques available for micro- and nano-scale mechanical characterization have exploded in the last few decades. From further development of the scanning ...

Bioingegneria

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking (TXL) for Myopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy include photochemical cross-linking and tissue cross-linking (TXL) methods. Such methods for inducing mechanical tissue property changes are being employed to the cornea in corneal thinning (mechanically weakened) disorders such as keratoconus as well as the sclera in progressive myopia, where thinning and weakening of the posterior sclera occurs and likely contributes to axial elongation. The primary target proteins for such tissue strengthening are fibrillar collagens which constitute the great majority of dry weight proteins in the cornea and sclera. Fortuitously, fibrillar collagens are the main source of second harmonic generation signals in the tissue extracellular space. Therefore, modifications of the collagen proteins, such as those induced through cross-linking therapies, could potentially be detected and quantitated through the use of second harmonic generation microscopy (SHGM). Monitoring SHGM signals through the use of a laser scanning microscopy system coupled with an infrared excitation light source is an exciting modern imaging method that is enjoying widespread usage in the biomedical sciences. Thus, the present study was undertaken in order to evaluate the use of SHGM microscopy as a means to measure induced cross-linking effects in ex vivo rabbit sclera, following an injection of a chemical cross-linking agent into the sub-Tenon&#39;s space (sT), an injection approach that is standard practice for causing ocular anesthesia during ophthalmologic clinical procedures. The chemical cross-linking agent, sodium hydroxymethylglycinate (SMG), is from a class of cosmetic preservatives known as formaldehyde releasing agents (FARs). Scleral changes following reaction with SMG resulted in increases in SHG signals and correlated with shifts in thermal denaturation temperature, a standard method for evaluating induced tissue cross-linking effects.

Second Harmonic Generation Signals in Rabbit Sclera As a Tool for Evaluation of Therapeutic Tissue Cross-linking TXL for Myopia

This protocol describes techniques for evaluating chemical cross-linking of the rabbit sclera using&#160;second harmonic generation imaging and differential scanning calorimetry.

Seconda generazione di armoniche segnali coniglio sclera come strumento per la valutazione del tessuto terapeutico reticolazione (TXL) per la miopia

Methods to strengthen tissue by introducing chemical bonds (non-enzymatic cross-linking) into structural proteins (fibrillar collagens) for therapy ...

Full-Circle Cauterization of Limbal Vascular Plexus for Surgically Induced Glaucoma in Rodents

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic nerve and retinal ganglion cell (RGC) degeneration, resulting in visual dysfunction by interrupting the transmission of visual information from the eye to the brain. Elevated intraocular pressure is the most important risk factor; thus, several models of ocular hypertension have been developed in rodents by either genetic or experimental approaches to investigate the causes and effects of the disease. Among those, some limitations have been reported such as surgical invasiveness, inadequate functional assessment, requirement of extensive training, and highly variable extension of retinal damage. The present work characterizes a simple, low-cost, and efficient method to induce ocular hypertension in rodents, based on low-temperature, full-circle cauterization of the limbal vascular plexus, a major component of aqueous humor drainage. The new model provides a technically easy, noninvasive, and reproducible subacute ocular hypertension, associated with progressive RGC and optic nerve degeneration, and a unique post-operative clinical recovery rate that allows in vivo functional studies by both electrophysiological and behavioral methods.

The goal of this protocol is to characterize a novel model of glaucomatous neurodegeneration based on 360&#176; thermic cauterization of limbal vascular plexus, inducing subacute ocular hypertension.

Cauterizzazione a cerchio completo del plesso vascolare limbare per il glaucoma indotto chirurgicamente nei roditori

Glaucoma, the second leading cause of blindness worldwide, is a heterogeneous group of ocular disorders characterized by structural damage to the optic ...

Neuroscienze

A Tissue Clearing Method for Neuronal Imaging from Mesoscopic to Microscopic Scales

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging from neural circuits to subcellular neuronal structures are visualized in mouse brain slices optically cleared with ScaleSF. This clearing method is a modified version of ScaleS and is a hydrophilic tissue clearing method for tissue slices that achieves potent clearing capability as well as a high-level of preservation of fluorescence signals and structural integrity. A customizable three dimensional (3D)-printed imaging chamber is designed for reliable mounting of cleared brain tissues. Mouse brains injected with an adeno-associated virus vector carrying enhanced green fluorescent protein gene were fixed with 4% paraformaldehyde and cut into slices of 1-mm thickness with a vibrating tissue slicer. The brain slices were cleared by following the clearing protocol, which include sequential incubations in three solutions, namely, ScaleS0 solution, phosphate buffer saline (&#8211;), and ScaleS4 solution, for a total of 10.5&#8211;14.5 h. The cleared brain slices were mounted on the imaging chamber and embedded in 1.5% agarose gel dissolved in ScaleS4D25(0) solution. The 3D image acquisition of the slices was carried out using a confocal laser scanning microscope equipped with a multi-immersion objective lens of a long working distance. Beginning with mesoscopic neuronal imaging, we succeeded in visualizing fine subcellular neuronal structures, such as dendritic spines and axonal boutons, in the optically cleared brain slices. This protocol would facilitate understanding of neuronal structures from circuit to subcellular component scales.

The protocol provides a detailed method of neuronal imaging in brain slice using a tissue clearing method, ScaleSF. The protocol includes brain tissue preparation, tissue clarification, handling of cleared slices and confocal laser scanning microscopy imaging of neuronal structures from mesoscopic to microscopic levels.

Un metodo di compensazione dei tessuti per l'imaging neuronale dalle scale mesoscopiche a quelle microscopiche

A detailed protocol is provided here to visualize neuronal structures from mesoscopic to microscopic levels in brain tissues. Neuronal structures ranging ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

Inseguimento In Vivo dei timociti nell'alloggiamento anteriore dell'occhio di microscopia a scansione Laser in tempo reale

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Biologia

Preparazione e imaging di montature intere per lenti oculari murine

Whole Mount Imaging to Visualize and Quantify Peripheral Lens Structure, Cell Morphology, and Organization

The ocular lens is a transparent flexible tissue that alters its shape to focus light from different distances onto the retina. Aside from a basement membrane surrounding the organ, called the capsule, the lens is entirely cellular consisting of a monolayer of epithelial cells on the anterior hemisphere and a bulk mass of lens fiber cells. Throughout life, epithelial cells proliferate in the germinative zone at the lens equator, and equatorial epithelial cells migrate, elongate, and differentiate into newly formed fiber cells. Equatorial epithelial cells substantially alter morphology from randomly packed cobble-stone-shaped cells into aligned hexagon-shaped cells forming meridional rows. Newly formed lens fiber cells retain the hexagonal cell shape and elongate toward the anterior and posterior poles, forming a new shell of cells that are overlaid onto previous generations of fibers. Little is known about the mechanisms that drive the remarkable morphogenesis of lens epithelial cells to fiber cells. To better understand lens structure, development, and function, new imaging protocols have been developed to image peripheral structures using whole mounts of ocular lenses. Here, methods to quantify capsule thickness, epithelial cell area, cell nuclear area and shape, meridional row cell order and packing, and fiber cell widths are shown. These measurements are essential for elucidating the cellular changes that occur during lifelong lens growth and understanding the changes that occur with age or pathology.

Autore in primo piano: Esame morfometrico approfondito e quantificazione della struttura nativa della lente utilizzando l'imaging a montatura intera 

The present protocols describe novel whole mount imaging for the visualization of peripheral structures in the ocular lens with methods for image quantification. These protocols can be used in studies to better understand the relationship between lens microscale structures and lens development/function.

Imaging a montaggio completo per visualizzare e quantificare la struttura periferica del cristallino, la morfologia e l'organizzazione cellulare

The ocular lens is a transparent flexible tissue that alters its shape to focus light from different distances onto the retina. Aside from a basement ...

Utilizzando una tecnica di laminazione per l'esecuzione di Microscopia confocale della sclera umana

Riepilogo

Esplora Altri Video

Capitoli in questo video

Microscopia corneale confocale: A Novel tecnica non invasiva per quantificare Piccole Fibre Patologia neuropatie periferiche

Rendering tridimensionale e l'analisi di Immunolabeled, chiarito reti vascolari Villous placentare umani

Microscopia di luce-foglio per visualizzazione tridimensionale delle cellule immuni umane

Microscopia confocale e a forza atomica (Conpokal) quasi simultanea su cellule vive

Seconda generazione di armoniche segnali coniglio sclera come strumento per la valutazione del tessuto terapeutico reticolazione (TXL) per la miopia

Cauterizzazione a cerchio completo del plesso vascolare limbare per il glaucoma indotto chirurgicamente nei roditori

Un metodo di compensazione dei tessuti per l'imaging neuronale dalle scale mesoscopiche a quelle microscopiche

Inseguimento In Vivo dei timociti nell'alloggiamento anteriore dell'occhio di microscopia a scansione Laser in tempo reale

Imaging a montaggio completo per visualizzare e quantificare la struttura periferica del cristallino, la morfologia e l'organizzazione cellulare

Imaging a montaggio completo per visualizzare e quantificare la struttura periferica del cristallino, la morfologia e l'organizzazione cellulare