Ringraziamo P. Verrijzer, J. Johansen H., Okhura per gli anticorpi e A. Debec, i centri di borsa bloomington e vienna per le mosche. Grazie a C. Jackson per suggerimenti e discussioni stimolanti.

1. Raccolta testes Drosophila
NOTA: I giovani maschi (0-15 ore dopo l'eclosion) sono necessari per esaminare le cellule germinali diploidi (ad esempio spermatogonia e spermatociti).
<ol><li>Seleziona le mosche giovani il più possibile per ridurre al minimo le interferenze derivanti dallo sviluppo di spermatidi.</li>
	<li>Anestetizza le mosche con una breve esposizione all'anidride carbonica e si trasferisce su un fly pad14,15.</li>
	<li>Al microscopio sezionato (ingrandimento 10x), utilizzare le flessione per rimuovere la testa.</li>
	<li>Trasferire da 5 a 10 mosche decapitate a 100 μL del tampone di Ringer (183 mM KCI, 47 mM NaCl, 10 mM Tris-HCI, 1 mM EDTA, inibitore proteico completo, pH 6,8) su uno scivolo di vetro rivestito di silicone su un supporto di sfondo nero.</li>
	<li>Con il microscopio sezionato con un ingrandimento di 40x, utilizzare un paio di forcep numero 5 per tenere una mosca tra il torace e l'addome e, con altre forcep, allontanare l'addome dal torace. Isolare rapidamente i tester e i tessuti adiacenti dalla carcassadi mosca 15 e posizionare in una nuova goccia del buffer di Ringer sullo stesso scivolo.</li>
	<li>Una volta che tutti i teste sono pronti, pulire i teste dai tessuti rimanenti (ghiandola accessoria e genitali esterni).</li>
	<li>Rimuovere il buffer in eccesso dalla goccia contenente i teste sezionati. In genere, utilizzare una pipetta p200 con una punta da 10 μL montata sopra una punta da 200 μL. Ciò consente la rimozione dell'eccesso di liquido attraverso una piccola apertura. Posizionare una goccia del 4% di paraformaldeide fissativa sopra la goccia contenente i tester sezionati, quindi trasferire rapidamente i tester su un tubo contenente soluzione fissante fresca.</li>
</ol>2. Fissazione della paraformaldeide
<ol><li>Preparare 2 ml di soluzione fissativa poco prima dell'uso: 600 μL di paraformaldeide al 4% diluita in salina tamponata di fosfato (PBS) con 30 μL di NP40 20% più 1200 μL di eptano in un tubo di microcentrifugo siliconato da 2 ml. Utilizzare il 4% di paraformaldeide in PBS preparata in aliquote 600 μL e conservata a -20°C. Attenzione: Eptano e paraformaldeide sono tossici e devono essere maneggiati in una cappa aspirante. Smaltirli secondo il regolamento dell'istituto ospitante.</li>
	<li>Trasferire i tester utilizzando una micropipetta p200 con un'ampia punta di apertura alla soluzione PFA/NP40/eptano. Agitare il tubo su e giù per 30 s a mano. Continuare a fissare i tester su un miscelatore rotativo a temperatura ambiente per 30 minuti.</li>
	<li>Arrestare il miscelatore rotante e posizionare il tubo in un rack per tubi per consentire la separazione di fase e per i tester di depositarsi sul fondo del tubo.</li>
	<li>Rimuovere tutto l'eptano e la soluzione fissante e risciacquare rapidamente i tester tre volte con 1x PBS.</li>
	<li>Trasferire in un singolo tubo pulito da 200 μL. Eseguire tutte le incubazioni in questi piccoli tubi per ridurre al minimo le quantità di anticorpi impiegati.</li>
	<li>Rimuovere il buffer in eccesso utilizzando una micropipetta da 200 μL con una punta P200 e una punta P10 attaccata. Fare attenzione a non aspirare teste.</li>
	<li>Conservare i test in 1x PBS sul ghiaccio fino a quando tutti i campioni non sono pronti.</li>
</ol>3. Colorazione anticorpale in soluzione
<ol><li>Scartare il PBS e lasciare i tester nello 0,3% di PBS-T per 30 minuti a temperatura ambiente per permeabilizzare le membrane cellulari.</li>
	<li>Pre-incubare nello stesso tampone più il 5% di siero di capra normale (NGS) per 1 h.</li>
	<li>Aggiungere 200 μL dell'anticorpo primario diluito nello 0,3% pbs-t più 5% NGS(Tabella dei materiali).</li>
	<li>Incubare l'anticorpo primario durante la notte a 4 °C (o temperatura ambiente) con delicata agitazione su un rocker.</li>
	<li>Lavare 3 volte in 0,3% PBS-T per 15 minuti su un rocker a temperatura ambiente. Lasciare che i tester si sistemino sul fondo del tubo per gravità.</li>
	<li>Aggiungere 200 μL di anticorpo secondario diluito 1:500 dalla serie coniugata DyLight.</li>
	<li>Incubare l'anticorpo secondario in una camera scura per 4 ore a temperatura ambiente.</li>
	<li>Lavare in 0,3% PBS-T per 15 minuti su un rocker a temperatura ambiente. Quindi ripetere con 0,2% PBS-T e 0,1% PBS-T, ogni volta per 30 minuti su un rocker a temperatura ambiente. Fare un lavaggio finale in 1x PBS per 30 minuti per rimuovere il detergente.</li>
	<li>Aggiungere 180 μL di soluzione DAPI a 1 μg/mL in 1x PBS per 15 min. La soluzione stock è di 10 mg/mL in H2O. Evitare di fare soluzione stock in PBS, che può ridurre il segnale di colorazione DAPI sulla cromatina diffusa negli spermatociti più grandi.</li>
	<li>Lavare 3 volte per 5 minuti ciascuno.</li>
	<li>Utilizzare una penna barriera idrofobica per disegnare un cerchio su uno scivolo pulito.</li>
	<li>Aspirare i teste e metterli sulla diapositiva. Il preri risciacquo della punta di apertura ampia con PBS-T dello 0,1% impedisce ai tester di attaccarsi alla punta. Rimuovere il PBS in eccesso.</li>
	<li>Aggiungere una goccia (circa 100 μL) di Citifluor AF1.</li>
	<li>Posizionare delicatamente una coverlip di vetro sul tessuto, facendo attenzione a evitare di intrappolare bolle d'aria.</li>
	<li>Sigillare il coverslip allo scivolo utilizzando smalto chiaro.</li>
	<li>Osservare le diapositive al microscopio.</li>
</ol>4. Analisi di colocalizzazione
<ol><li>Acquisire immagini confocali. Abbiamo usato un microscopio confocale Zeiss LSM 710 (olio apocromatico a piano 63x) con una risoluzione z di 0,5 o 1 μm.</li>
	<li>Selezionare celle diverse da tester indipendenti.</li>
	<li>Importare immagini nel software di elaborazione (ad esempio, Imaris).</li>
	<li>Definire il nucleo mediante segmentazione manuale per escludere i nuclei vicini. Utilizzare il software Coloc; raccoglie i coefficienti di correlazione di Pearson (PCC) all'interno dell'intero volume tra 2 fluorofori.</li>
</ol>

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Gli autori non hanno nulla da rivelare.

Questo protocollo fornisce un metodo per l'immunoetichettatura di tester di Drosophila adulti a montaggio intero. Come riportato in altre procedure devono essere utilizzati giovani maschi (abbiamo anche ridotto l'età a meno di 20 ore dopo l'eclosion) per ridurre al minimo la presenza di spermatozoi in via di sviluppo, che rischia di ostruire gli spermatociti. La dissezione deve essere rapida e la diluizione degli anticorpi deve essere regolata per ottimizzare il rapporto segnale-rumore21,</s...

I test di Drosophila sono un modello prezioso per lo studio dell'architettura nucleare. Nelle cellule spermatogoniche mitotiche, il volume nucleare è in gran parte occupato dalla cromatina. Queste cellule subiscono quattro cicli di divisione, producendo una cisti di 16 spermatociti primari. Nei giorni successivi, gli spermatociti passano attraverso 6 stadi di sviluppo (S1-S6), aumentando notevolmente di volume, approssimativamente 20 volte1,2. Cambiano anche la loro morfologia nucleare. Il contorno del nucleo, rivelato dal lamina Dm0, diventa irregolare e mostra profonde invaginazioni1,3. Questo è accompagnato da ampi cambiamenti di espressione genica e modifiche nell'organizzazione della cromatina1,4,5,6,7. I cromosomi interfase sono ben definiti nello stadio S4-S5, formando tre masse distinte corrispondenti ai 2 principali autosomi bivalenti e alla coppia X-Y agganciata al nucleolo.
Mad1 è stato originariamente isolato come componente chiave del checkpoint mitotico (esaminato in8). Nell'interfase, è descritto come situato principalmente all'involucro nucleare ma anche nel nucleoplasma9,10,11. In Drosophila testis, abbiamo riferito che Mad1 è prominente nel nucleoplasma, intimamente associato alle due principali masse di cromatina autosomica e in misura minore alla cromatina XY. Abbiamo definito questa struttura associata alla cromatina come MINT (Mad1-containing IntraNuclear Territory)12. Altre quattro proteine erano associate ai TT negli spermatociti: la nucleoporina Mtor/Tpr, la peptidasi Ulp1 suMO, la proteina del checkpoint mitotico Mad2 e una subunità di un complesso simile a Policomb Raf212.
Per completare la descrizione dei MINT, dovevamo usare gli anticorpi e ci siamo trovati di fronte ai problemi tecnici generalmente incontrati con l'immunostaining nel testicolo: una scarsa permeabilità con conseguente significativa non uniformità di colorazione nella profondità del testicolo, in particolare nei grandi spermatociti. Ipreparati s annullati aumentavano l'accesso agli anticorpi ma portavano a immagini compresse, limitando le analisi 3D e impedendo la misurazione della fluorescenza quantitativa, compresa la colocalizzazione. Il problema della penetrazione degli anticorpi potrebbe anche essere affrontato da agenti permeabilizzanti come lo 0,3% di Na deossicholato13, ma questa procedura non ha prodotto nelle nostre mani risultati riproducibili. Poiché abbiamo eseguito molti esperimenti di co-etichettatura, abbiamo cercato di migliorare il protocollo di permeabilizzazione. La combinazione di 1% NP40 più eptano ha portato a una maggiore riproducibilità, in particolare nello studio dei grandi spermatociti.
Questo protocollo esegue la fissazione e l'immunosocienza dei test in sospensione, migliorando la qualità del campione e migliorando la riproducibilità e rendendolo adatto per la microscopia confocale. Abbiamo usato il protocollo per definire meglio le relazioni spaziali di alcune proteine dell'involucro nucleare con le strutture nucleoplasmatiche dei grandi spermatociti. Questo metodo consentirà di esaminare meglio i cambiamenti che accompagnano lo sviluppo degli spermatociti, ad esempio i cambiamenti strutturali dinamici del nucleo, tra cui cromatina, nucleoplasma e pori nucleari.

<table border="0" cellpadding="0" cellspacing="0" style="width:1005px;" width="1005">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Drosophila srains</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td style="width:505px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">ketelGFP y; ketelGFP/y+CyO</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>Gift from A. Debec (Institut Jacques Monod, UMR 7592, Paris, France)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Mad1-GFP</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>J Cell Sci. 2011 May 15;124(Pt 10):1664-71.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Mtor RNAi&#160;</td>
			<td style="width:129px;">VDRC</td>
			<td style="width:93px;">ID 110218</td>
			<td>Vienna Drosophila RNAi Center</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Materials</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:277px;">DAPI&#160;</td>
			<td style="width:129px;">Thermo</td>
			<td>D1306</td>
			<td>Stock solution must be prepared in H2O</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Dumont # 5 Fine Forseps&#160;</td>
			<td style="width:129px;">Fine Science Tools</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MBP Wide Bore Pipette Tips</td>
			<td style="width:129px;">Fisherbrand</td>
			<td>11917744</td>
			<td>Wide aperture tip</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermo Scientific&#160;0.2 mL Individual Tube</td>
			<td style="width:129px;">Thermo</td>
			<td>AB-0620</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 ml micro centrifuge tubes&#160;</td>
			<td style="width:129px;">Sigma</td>
			<td>T3531-200EA&#160;</td>
			<td>Siliconized 2ml tube</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Citifluor AF1</td>
			<td style="width:129px;">Citifluor</td>
			<td>AF1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dakopen&#160;</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:93px;">Z377821-1EA&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum (NGS)</td>
			<td style="width:129px;">Sigma</td>
			<td>NS02L-1ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde 4%</td>
			<td style="width:129px;">Sigma</td>
			<td style="width:93px;">P6148-500G&#160;</td>
			<td>Paraformaldehyde (4%) dissolved in PBS 1X&#160;; aliquot by 600 ml&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS 1X</td>
			<td style="width:129px;">Thermo</td>
			<td>14190094</td>
			<td>DPBS, no calcium, no magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.1% (0.2 or 0.3%) PBS-T&#160;</td>
			<td style="width:129px;"></td>
			<td></td>
			<td>PBS 1X&#160; containing 0.1% (0.2 % or 0.3%) Triton X-100</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100, for molecular biology,&#160;</td>
			<td style="width:129px;">Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP booster</td>
			<td style="width:129px;">Chromotek</td>
			<td>gba488</td>
			<td>1/200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mouse anti-Mtor</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td style="width:505px;">1/40 gift from J. Johansen, Iowa State University</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat anti-Nup62</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td style="width:505px;">1/200 gift from H. Ohkura (University of Edinburgh, UK</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guinea-pig anti-Ulp1</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Rabbit anti-Raf2</td>
			<td style="width:129px;"></td>
			<td style="width:93px;"></td>
			<td>1/200 gift from C. P. Verrijzer, Erasmus University, Rotterdam</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DyLight conjugated series&#160;</td>
			<td style="width:129px;">Thermo</td>
			<td style="width:93px;"></td>
			<td>1/500 Donkey anti-(guinea-pig, mouse,&#160; rabbit or rat) as second antibodies</td>
		</tr>
	</tbody>
</table>

immunostaining of whole-mount drosophila testes for 3d confocal analysis of large spermatocytes

I test di Drosophila sono un potente sistema modello per lo studio dei processi biologici tra cui la biologia delle cellule staminali, l'architettura nucleare, la meiosi e lo sviluppo dello sperma. Tuttavia, l'immunoetichettatura dell'intero testicolo drosophila è spesso associata a una significativa non uniformità di colorazione dovuta alla penetrazione degli anticorpi. I preparati schiacciati superano solo parzialmente il problema poiché diminuisce la qualità 3D delle analisi. Qui, descriviamo un protocollo a montaggio intero usando NP40 ed eptano durante la fissazione insieme all'immunoetichettatura nei mezzi liquidi. Preserva il volume adatto alla microscopia confocale insieme a etichette riproducibili e affidabili. Mostriamo diversi esempi di ricostituzione 3D dei nuclei di spermatociti da sezioni confocali. La riproducibilità intra- e inter-testes consente la quantificazione 3D e il confronto della fluorescenza tra singole cellule di diversi genotipi. Abbiamo utilizzato diversi componenti della struttura MINT intranucleare (Mad1-containing Intra Nuclear Territory) e due componenti associati al complesso dei pori nucleari per illustrare questo protocollo e le sue applicazioni sulle più grandi cellule del testicolo, gli spermatociti S4-S5.

Il principale ostacolo all'ottenimento di un'adeguata colorazione dei test di Drosophila è la limitata penetrabilità degli anticorpi, che è stata parzialmente risolta utilizzando preparati schiacciati ma a costo di indurre una restrizione delle analisi 3D (ad esempio rappresentazione 3D o misura della colocalizzazione). La procedura consente un'etichettatura uniforme e preserva il volume di tutte le cellule del testicolo. Qui abbiamo focalizzato l'illustrazione del metodo sulla rappresentazione 3D degli spermatociti S...

Watch this Scientific Journal Video about Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes at JoVE.com

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Presentiamo qui un protocollo migliorato per la preparazione a montaggio intero e l'immunostaining dei tester Drosophila, adatto per la microscopia confocale e che consente anche un'etichettatura riproducibile e affidabile. Illustriamo questo protocollo attraverso la rappresentazione 3D e la quantificazione degli esperimenti di colocalizzazione sui grandi spermatociti S4-S5.

Immunostaining dei test di Drosophila a montaggio intero per l'analisi confocale 3D di grandi spermatociti

Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm ...

immunostaining-whole-mount-drosophila-testes-for-3d-confocal-analysis

Research

JoVE Journal

Biology

Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

4.1K Views.  Université de Paris, F 75006, Paris, France. Presentiamo qui un protocollo migliorato per la preparazione a montaggio intero e l'immunostaining dei tester Drosophila, adatto per la microscopia confocale e che consente anche un'etichettatura riproducibile e affidabile. Illustriamo questo protocollo attraverso la rappresentazione 3D e la quantificazione degli esperimenti di colocalizzazione sui grandi spermatociti S4-S5.

Video: Immunostaining of Whole-Mount Drosophila Testes for 3D Confocal Analysis of Large Spermatocytes

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Doppia Monte intero ibridazione in situ di embrioni di pollo primi

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Ricerca

Biologia

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Metodo per la colorazione intero anticorpi Monte a Chick

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish intero monte ad alta risoluzione doppia fluorescente In Situ Ibridazione

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Batch Immunostaining for Large-Scale Protein Detection in the Whole Monkey Brain

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the expression pattern of a target protein across the entire brain are relatively easy to answer when using IHC in small brains, such as those of rodents. However, answering the same questions in large and convoluted brains, such as those of primates presents a number of challenges. Here we present a systematic approach for immunodetection of target proteins in an adult monkey brain. This approach relies on the tissue embedding and sectioning methodology of NeuroScience Associates (NSA) as well as tools developed specifically for batch-staining of free-floating sections. It results in uniform staining of a set of sections which, at a particular interval, represents the entire brain. The resulting stained sections can be subjected to a wide variety of analytical procedures in order to measure protein levels, the population of neurons expressing a certain protein.

Large-scale immunodetection of target proteins across the entire primate brain is possible by employing novel tissue embedding and sectioning methods combined with the use of creative apparatus for batch staining of multiple free-floating sections at a given time.

Immunostaining lotto per la grande rivelazione di proteine ​​nel cervello della scimmia Whole

Immunohistochemistry (IHC) is one of the most widely used laboratory techniques for the detection of target proteins in situ. Questions concerning the ...

Isolation of Drosophila melanogaster Testes

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline interactions. Testes are typically isolated from adult males 0-3 days after eclosion from the pupal case. The testes of wild-type flies are easily distinguished from other tissues because they are yellow, but the testes of white mutant flies, a common genetic background for laboratory experiments are similar in both shape and color to the fly gut. Performing dissection on a glass microscope slide with a black background makes identifying the testes considerably easier. Testes are removed from the flies using dissecting needles. Compared to protocols that use forceps for testes dissection, our method is far quicker, allowing a well-practiced individual to dissect testes from 200-300 wild-type flies per hour, yielding 400-600 testes. Testes from white flies or from mutants that reduce testes size are harder to dissect and typically yield 200-400 testes per hour.

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testes can be rapidly and efficiently isolated from adult males using dissecting needles. With practice, one can readily isolate in one or two days an amount of testes sufficient for the analysis of DNA or RNA by high throughput sequencing or more traditional molecular biology methods or of protein for antibody- or enzyme-based assays.

Isolamento di Drosophila melanogaster Testicoli

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Tutto il monte In Situ</emIbridazione> di E8.5 a E11.5 embrioni di topo

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Tutta la Monte RNA fluorescente<em&gt; In situ</em&gt; L&#39;ibridazione di<em&gt; Drosophila</em&gt; Embrioni

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

L&#39;analisi citologica della spermatogenesi: Preparati Live e fisse di<em&gt; Drosophila</em&gt; Testicoli

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to Detect Activated Caspase 3

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis in zebrafish embryos. The whole-mount analysis provides spatial information in regard to tissue specificity of apoptosing cells, although sectioning and/or colabeling is ultimately required to pinpoint the exact cell types undergoing apoptosis. The whole-mount Casp3 assay is optimized for analysis of fixed embryos between the 4-cell stage and 32 hr-post-fertilization and is useful for a number of applications, including analysis of zebrafish mutants and morphants, overexpression of mutant and wild-type mRNAs, and exposure to chemicals. Compared to acridine orange staining, which can identify apoptotic cells in live embryos in a matter of hours, Casp3 and TUNEL assays take considerably longer to complete (2-4 days). However, because of the dynamic nature of apoptotic cell formation and clearance, analysis of fixed embryos ensures accurate comparison of apoptotic cells across multiple samples at specific time points. We have also found the Casp3 assay to be superior to analysis of apoptotic cells by the whole-mount TUNEL assay in regard to cost and reliability. Overall, the Casp3 assay represents a robust, highly reproducible assay in which to analyze apoptotic cells in early zebrafish embryos.

Certain genetic perturbations or exposure to toxins can disrupt normal developmental processes leading to death of specific cell types. The analysis of activated Caspase 3 by whole-mount immunofluorescence in zebrafish embryos reveals stage- and tissue-specific localization of cells specifically undergoing apoptosis.

Analisi di apoptosi in embrioni di zebrafish da Whole-mount immunofluorescenza per rilevare caspasi 3 attivata

Whole-mount immunofluorescence to detect activated Caspase 3 (Casp3 assay) is useful to identify cells undergoing either intrinsic or extrinsic apoptosis ...

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 ...