Name: visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish
Uploaded: 2016-12-21T13:00:00.000+00:00
Duration: 7 min 19 s
Description: Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Ringraziamo il Prof. Christoph Englert e il Prof. Didier Stainier per regalare il Tg (wt1b: GFP) LI1 e Tg (kdrl: EGFP) s843 ceppi, rispettivamente, e il meccanismo di Taiwan Zebrafish Nucleo per la fornitura di Tg (kdrl: mCherry) CI5. Questo studio è stato sostenuto da sovvenzioni dal Ministero della Scienza e della Tecnologia (96-2628-B-029-002-MY3, 101-2313-B-029-001, 102-2628-B-029-002-MY3 di Taiwan, 102- 2321-B-400-018).

Etica Dichiarazione: Tutte le procedure sperimentali su zebrafish sono stati approvati dal Comitato di Cura e uso istituzionale degli animali di Tunghai University (IRB Soddisfazione NO 101-12.) E realizzato in conformità con le linee guida approvate. 1. Soluzioni azioni 3β-Hsd enzimatico attività di colorazione <ol><li> Preparare trans-dehydroandrosterone [10 mg / ml in dimetilsolfossido (DMSO)]. </li><li> Preparare β-nicotinammide adenina dinucleotide idrato (1,2 mg / ml in tampone fosfato 0,1 M, pH 7,2). </li><li> Preparare nicotinamide (vitamina B3, 50 mg / ml in H 2 O). </li><li> Preparare 4-nitro tetrazolio blu (50 mg / ml nel 70% dimetilformammide). </li><li> Aliquota tutti questi componenti in tubi microcentrifuga e conservare a -20 ° C. NOTA: Nella nostra esperienza, ripetuti di congelamento e scongelamento delle aliquote di tutte le soluzioni di cui sopra non influenzano l&#39;intensità della 3β-Hsd attività di colorazione. </li></ol> 2. Whole-mount 3β-Hsd attività di colorazione NOTA: L&#39;attività enzimatica 3β-HSD è rilevabile in embrioni di zebrafish come già 28 ore dopo la fecondazione (HPF) quando coltivate a 28,5 ° C, che è coerente con l&#39;inizio di espressione dell&#39;mRNA di hsd3b1 2,7. Il tutto il montaggio 3β-Hsd protocollo attività colorazione in questo studio si basa su un metodo precedentemente descritto 8 ed era determinato ad avere successo nel zebrafish in via di sviluppo fino a 7 giorni dopo la fecondazione 9. Per analizzare il microambiente vascolare del tessuto steroidea interrenale, tutto il montaggio 3β-Hsd attività colorazione deve essere applicato a linee transgeniche che esprimono fluorescenza specifica-endotelio, come ad esempio Tg (kdrl: EGFP) s843 14 e Tg (kdrl: mCherry) CI5 15 . Per analizzare il tessuto steroidogenica interrenale considerando lo sviluppo del rene zebrafish, 3β-Hsd attività colorazione può essere applied al Tg (wt1b: GFP) pesci LI1 16, dove si delinea la formazione del glomerulo e pronephric tubuli. <ol><li> Trattare il zebrafish sviluppo incubandoli in 0,03% phenylthiourea in acqua uovo (0,06 mg / L barriera sale in acqua deionizzata), partendo da una fase compresa tra 12 e 24 HPF, per inibire la formazione di pigmento. </li><li> Fix embrioni dechorionated o larve in (A) 4% paraformaldeide (PFA) in soluzione salina tamponata con fosfato (PBS) supplementato con 0,1% Tween 20 (PFAT) per 1 ora a temperatura ambiente (RT) o (B) 2% PFAT notte a 4 ° C o per 2 ore a 4 ° C, seguita da 4 ore a RT. Attenzione: PFA è tossico per inalazione e per contatto con la pelle. E &#39;distruttivo per la pelle, le mucose, gli occhi e le vie respiratorie superiori. </li><li> Lavare il embrioni 4x per 10 minuti con PBS integrato con 0,1% di Tween 20 (PBST) a temperatura ambiente. NOTA: E &#39;fondamentale per non secca i campioni, mentre la modifica delle soluzioni di lavaggio. </li><li> Appena preparare la STAsoluzione ining prima delle reazioni (Tabella 1). Preparare, vortice, e componenti per centrifughe di A e B in tubi separati. NOTA: L&#39;aggiunta di tutti i componenti direttamente in un unico tubo potrebbe causare gravi precipitazioni. </li><li> Aggiungere l&#39;intera soluzione della parte A per l&#39;intera soluzione di parte B, mescolare bene per preparare la soluzione colorante, e subito distribuire 1 ml in ogni provetta da microcentrifuga contenente embrioni fissi e lavati. </li><li> Avvolgere i tubi microcentrifuga con un foglio di alluminio per proteggerli dalla luce e luogo sia su un rotatore o di una piattaforma oscillante per gentile agitazione a temperatura ambiente. </li><li> Empiricamente determinare il tempo di reazione per il monitoraggio dei segnali cromogenici mediante microscopia. precipitazioni lievi si svilupperà nel corso del tempo; tuttavia, essi non interferiscano con il processo di reazione. NOTA: per gli embrioni fissati a 28 HPF, colorazione a 25 ° C per 4 ore genera un segnale chiaro al tessuto interrenale. </li><li> Arrestare la reazione lavando 4x per 10 min in PBST. iof è richiesta alcuna ulteriore immunoistochimica (IHC) analisi, post-correggere gli embrioni colorate con 4% PFAT per 60 minuti a temperatura ambiente. Altrimenti, memorizzare gli embrioni lavati a 4 ° C fino all&#39;utilizzo. </li><li> Per tutto il montaggio microscopia, cancellare gli embrioni macchiati, post-fisso, e lavato con il 50% glicerolo in PBS, orientarli con il lato dorsale rivolto verso l&#39;alto su una diapositiva, e osservare con un microscopio verticale sotto illuminazione in campo chiaro. NOTA: per creare un&#39;immagine ad alta risoluzione della morfologia del tessuto interrenale, analisi dell&#39;embrione 3β-Hsd attività macchiato e deyolked TV a montaggio è raccomandato. Come il tessuto steroidogenica interrenale è posizionato proprio sopra il sacco vitellino, una ventrale analisi TV a monte del tessuto interrenale permette una osservazione diretta e chiara della morfologia interrenale 17. </li></ol> 3. Analisi IHC di 3β-HSD embrioni attività macchiati NOTA: Per analizzare il microambiente vascolare del steroitessuto dogenic, gli embrioni di attività macchiati 3β-HSD con un reporter fluorescenti transgenici specifici endotelio sono sottoposti ad trasversali vibratome sezionamento e la successiva analisi IHC 12,18. <ol><li> Post-fissaggio <ol><li> Post-rimuovere gli embrioni 3β-Hsd activity-tinto e lavato con 2% PFA integrato con 1% Triton X-100 per 1 ora a 4 ° C e lavare 4x per 10 min in PBS contenente 1% Triton X-100 (PBSTx) a RT. </li></ol></li><li> Incorporare <ol><li> Preparare 4% a basso punto di fusione agarosio sciogliendo l&#39;agarosio in PBS a 95 ° C, aliquota in tubi microcentrifuga, e conservare a 4-8 ° C. </li><li> Sciogliere un&#39;aliquota del 4% bassofondente agarosio a 70 ° C per 10 minuti, e quindi mantenere il un&#39;aliquota a 47 ° C. Incorpora ogni embrione nel fuso 4% a basso punto di fusione agarosio in un berretto distaccata di una provetta da 1,5 ml microcentrifuga che funge da stampo e lasciare raffreddare fino solido. </li></ol></li><li> vibratome sezionamento <ol><li> Attaccare un pezzo di nastro di carta per la p seccalatform del vibratome. </li><li> Rimuovere il blocco di agarosio indurito dallo stampo da una lama di rasoio. Applicare colla al nastro di carta sulla piattaforma, e fissare il blocco di agarosio sul nastro di carta, con il lato anteriore del campione verso l&#39;alto. Tagliare il blocco di agarosio a una forma trapezoidale prismatica, con circa 4 mm e 8 mm per lato delle faccette superiore ed inferiore, rispettivamente. </li><li> Risciacquare blocco agarosio campioni contenenti con PBS freddo integrato con 0,5% Triton X-100 (0,5% PBSTx) utilizzando un contagocce. </li><li> Tagliare il blocco di agarosio in 100 sezioni micron secondo il manuale di istruzioni del vibratome. Costantemente lavare il blocco di agarosio per evitare l&#39;essiccazione. Come ogni fetta viene tagliata, toglierlo dal vibratome utilizzando un ago G 26 della siringa, e trasferire in un posto piatto contenente freddo 0,5% PBSTx (500 microlitri / pozzetto). </li><li> Raccogliere le sezioni di tessuto di attività-positivo 3beta-Hsd controllando al microscopio dissezione, e trasferire le sezioni da un PIpunta Pette con la sua estremità cut-off (circa 1 mm di diametro interno, in apertura) in una piastra a 96 pozzetti contenente freddo 0,5% PBSTx (150 ml / pozzetto). I campioni di tessuto di solito dissociano dal blocco agarosio dopo questo passaggio. </li></ol></li><li> Permeabilizzazione e lavaggio <ol><li> Lavare le fette 6x per 15 minuti a 0,5% PBSTx a RT, 10 min in PBS, e 4x per 10 min in PBS integrato con 1% di siero albumina bovina / 1% DMSO / 0,1% Triton X-100 (PBDTx). </li></ol></li><li> Blocco <ol><li> Incubare le fette per 1 hr a 2% di siero fetale bovino (FCS) in PBDTx a RT. </li></ol></li><li> Reazione anticorpo primario <ol><li> Incubare le fette per 1,5 giorni in PBDTx contenente un anticorpo primario (ad esempio, policlonale di coniglio anti-fibronectina umana e topo anti-umano adesione focale chinasi) a 4 ° C. </li></ol></li><li> Lavaggio <ol><li> Lavare le fette 6x per 10 min in PBDTx a temperatura ambiente. </li></ol></li><li> Blocco <ol><li> Incubare le fette per 1 ora a 2% FCS in PBDTx a temperatura ambiente. </li></ol></li><li> Reazione anticorpo secondario <ol><li> Incubare le fette per 1,5 giorni in PBDTx contenenti un anticorpo secondario (fluoroforo coniugato capra anti-coniglio o anti-topo IgG) a 4 ° C. </li></ol></li><li> Lavaggio <ol><li> Lavare le fette 6x per 10 min in PBDTx a temperatura ambiente e 4x per 10 min in PBST a temperatura ambiente. NOTA: Per l&#39;analisi al microscopio confocale, cancellare i campioni con il 50% glicerolo in PBS. </li></ol></li></ol> 4. Analisi confocale <ol><li> Osservare l&#39;intero embrione monte e la sezione con un microscopio confocale dotato di 10X / 20X e 0,5 / 0,75 lente dell&#39;obiettivo. </li><li> Per la rilevazione simultanea di fluorescenza e la colorazione 3β-Hsd, impostare la modalità multitraccia come segue: 488 nm laser argon e 505-530 nm filtro passa-banda per il rilevamento di GFP; 543 nm laser a elio-neon e 560-615 nm filtro passa-banda per il rilevamento di mCherry; e trasmessa canale di luce per la rivelazione di 3β-Hsd colorazione.La dimensione del foro stenopeico è 1 unità Airey, e la risoluzione è 1.024 x 1.024 pixel. </li><li> Montare le pile z (con 6 intervalli micron) come proiezione 3D, e unire la proiezione e il campo luminoso, utilizzando il software incorporato confocale. </li></ol>

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The authors have no competing financial interests to declare.

La forza dell&#39;attività 3β-Hsd segnale aumentato nel corso della reazione. chiari segnali di attività 3β-HSD sono stati rilevati dopo 4 ore di reazione per fasi, dalla 28 in poi HPF. Tuttavia, la durata della reazione richiede determinazione empirica, a seconda dello scopo del test. Nei casi in cui la colorazione richiede elaborazione notturna, uno sfondo leggermente bluastra tende a svilupparsi sui campioni. Questo problema può essere superato aumentando la durata di fissaggio (ad esempio, 4 ore a 4% P...

La ghiandola surrenale, una componente fondamentale dell&#39;asse ipotalamo-ipofisi-surrene, secerne gli steroidi e coordina l&#39;omeostasi di steroidi e la risposta del corpo allo stress. La ghiandola surrenale comprende la corteccia esterna, che secerne steroidi in maniera specifica per la zona, e midollo interni, che sintetizza catecolamine. La ghiandola interrenale in teleostei è la controparte della ghiandola surrenale nei mammiferi ed è composto da cellule interrenale e cromaffini steroidogenici, che sono equivalenti funzionali della corteccia surrenale e midollare, rispettivamente, 1-3. Studi condotti utilizzando il modello zebrafish hanno riferito che entrambe le linee cellulari steroidogenici e cromaffini sono formate da meccanismi molecolari e cellulari altamente simili a quelle dei mammiferi 1,2. Pertanto, il pesce zebra è un modello potenzialmente potente per lo studio di malattie genetiche, controllo neuroendocrino, e la biologia dei sistemi dell&#39;asse ipotalamo-ipofisi-surrene (interrenale). <p class = &quot;jove_content&quot;&gt; Nella ghiandola surrenale, 3β-Hsd catalizza la conversione del progesterone dal pregnenolone, 17α-idrossiprogesterone da 17α-hydroxypregnelolone, e androstenedione da deidroepiandrosterone 4,5. 3β-HSD è essenziale per biosynthesizing tutte le classi di steroidi ormonali, vale a dire il progesterone, glucocorticoidi, mineralcorticoidi, androgeni ed estrogeni,. I due umana 3β-Hsd isoenzimi HSD3B1 e HSD3B2 sono espressi in modo differenziale 6. HSD3B1 è espressa nella placenta e nei tessuti periferici, mentre HSD3B2 si esprime nella corteccia surrenale e gonadi. HSD3B1 umana e HSD3B2 sono co-ortologhi di hsd3b1 zebrafish, che si esprime al tessuto interrenale e gonadi adulti; hsd3b2 zebrafish è un gene maternamente espresso le cui trascrizioni scomparire prima organogenesi 7. Il protocollo di tutto il montaggio 3β-Hsd saggio di attività enzimatica per zebrafish è stato sviluppato modificando il metodo di Levy, uns descritto da Milano et al. , Su sezioni congelate di otto specie di teleostei 8. A causa della permeabilità dei tessuti e la trasparenza ottica del pesce zebra in via di sviluppo, tutto il montaggio 3β-Hsd istochimica può essere utilizzato con successo per l&#39;embrione zebrafish fisso e larve e specificamente delineare i tessuti interrenale differenziati. Questo test sensibile e rapida è stata applicata a vari mutanti e morphants dimostrano diversi tipi di dysmorphogenesis interrenale. L&#39;attività interrenale 3β-HSD è assente nell&#39;embrione se le specifiche del tessuto interrenale è interrotta attraverso una specifica atterramento del fattore di trascrizione Ff1b ed è diminuito come la differenziazione interrenale è influenzata da un atterramento del coregulator Ff1b Prox1 9,10. In particolare, l&#39;attività 3β-Hsd può essere rilevato in mutanti con gravi difetti nelle fasi iniziali, come ad esempio un occhio capocchia di spillo e strabismo, dove il 3β-Hsd istochimica delinea come la migrazione delle cellule interrenale è influenzata 11. La differenziazione del tessuto interrenale non è compromessa anche in completa assenza di sangue e vascolare. Pertanto, come forma di segnali endotelio-derivati in via di sviluppo dell&#39;organo interrenale può essere determinato 12,13. Nel complesso, questo saggio istochimico è stato utilizzato con successo per lo studio specificazione, differenziazione e migrazione delle cellule steroidogeniche nel modello zebrafish. Pertanto, dovrebbe essere un efficiente e uno strumento affidabile per eventuali schermi genetici o chimici di targeting disturbi degli organi surrenali e interrenale.

<table><tbody><tr><td>Confocal microscope</td><td>Carl Zeiss&amp;nbsp;</td><td>LSM510&amp;nbsp;</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>USB</td><td>US16374</td><td></td></tr><tr><td>Hyclone Fetal Calf Serum&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>SH30073</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide hydrate</td><td>Sigma</td><td>N1636</td><td></td></tr><tr><td>4-Nitro blue tetrazolium</td><td>Promega</td><td>S380C</td><td></td></tr><tr><td>Nusieve GTG</td><td>Lonza</td><td>50081</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Phenylthiourea</td><td>Sigma&amp;nbsp;</td><td>P7629</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P4417-100Tab</td><td></td></tr><tr><td>PYREX Spot Plate</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>Reef Salt</td><td>AZOO</td><td>AZ28001</td><td></td></tr><tr><td>trans-Dehydroandrosterone</td><td>Sigma</td><td>D4000</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000M</td><td></td></tr></tbody></table>

visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

Per determinare come i codevelops steroidogenici tessuto interrenale con il glomerulo renale pronephric e la sua vascolarizzazione nascente, il saggio di attività enzimatica 3β-HSD è stato eseguito sul doppio transgenici Tg (wt1b: GFP) LI1; Tg (kdrl: mCherry) CI5 embrione a 34 HPF (Figura 1). In questa fase, il tessuto steroidogenico activity-positive 3β-Hsd si trova alla linea mediana e caudale immediatamente glomeruli renali pronephri...

Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

La ghiandola interrenale in zebrafish è la controparte teleostei della ghiandola surrenale mammiferi. Questo protocollo introduce come eseguire la deidrogenasi 3-β-idrossisteroide (Δ 5-4 isomerasi; 3β-HSD) saggio di attività enzimatica, che rileva le cellule differenziate steroidogeniche nel zebrafish in via di sviluppo.

Visualizzare il Tissue interrenale Steroidogenic e la sua Vascolare Microenvironment in Zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

visualizing-interrenal-steroidogenic-tissue-its-vascular

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

6.9K Views.  Tunghai University.  La ghiandola interrenale in zebrafish è la controparte teleostei della ghiandola surrenale mammiferi. Questo protocollo introduce come eseguire la deidrogenasi 3-β-idrossisteroide (Δ 5-4 isomerasi; 3β-HSD) saggio di attività enzimatica, che rileva le cellule differenziate steroidogeniche nel zebrafish in via di sviluppo. 

Video: Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

Analisi dello sviluppo del rene Zebrafish con time-lapse imaging Utilizzando una dissezione microscopio dotato di sezionamento ottico

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Ricerca

Biologia dello sviluppo

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

Immunocolorazione fosfo-epitopi in Ciliated Organi di monte intero embrioni di zebrafish

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Chimica

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

La dissezione del larvale Zebrafish gonadica Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Imaging in vivo e quantitazione dell'ospite risposta angiogenica in Xenogrfts tumore zebrati

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Ricerca sul cancro

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Visualizzazione di cellule Multiciliated in Zebrafish attraverso un protocollo combinato di tutta Monte fluorescente ibridazione In Situ e immunofluorescenza

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Modelli di lesione di Zebrafish adulto per studiare gli effetti del Prednisolone nella rigenerazione di tessuto osseo

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

Quantificare le dimensioni del fegato nel pesce zebra Larval utilizzando la microscopia Brightfield

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Dissezione del rene Zebrafish adulti

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Biologia

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Lo studio diabete Through the Eyes of un pesce: Microdissection, visualizzazione e analisi della tg(fli:EGFP) adulto Vasculature retinico Zebrafish

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Medicina

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Preparazione di montaggio schermo per l&#39;osservazione e l&#39;analisi di zebrafish campioni macchiato da Whole Monte<em&gt; In situ</em&gt; Ibridazione

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...