Antikorlar için P. Verrijzer, J. Johansen H., Okhura ve sinekler için A. Debec, Bloomington ve Viyana stok merkezlerine teşekkür ederiz. Öneriler ve teşvik edici tartışmalar için C. Jackson'a teşekkürler.

1. Drosophila testis koleksiyonu
NOT: Genç erkekler (eklozyon sonrası 0-15 saat) diploid mikrop hücrelerini (spermatogonia ve spermatositler) incelemek için gereklidir.
<ol><li>Spermatidler geliştirmeden kaynaklanan paraziti en aza indirmek için mümkün olduğunca genç sinekleri seçin.</li>
	<li>Karbondioksite kısa bir süre maruz kalarak sinekleri uyuşturun ve bir sineklik pedine aktarın14,15.</li>
	<li>Bir diseksiyon mikroskobu (10x büyütme) altında, başı çıkarmak için tokmak kullanın.</li>
	<li>Siyah bir arka plan desteği üzerine silikon kaplı cam kaydırakta 5 ila 10 kafa kesilmiş sinekleri 100 μL Ringer tamponu (183 mM KCI, 47 mM NaCl, 10 mM Tris-HCI, 1 mM EDTA, komple protein inhibitörü, pH 6.8) aktarın.</li>
	<li>40x büyütmede mikroskobun parçalanmasıyla, toraks ile karın arasında bir sinek tutmak için bir çift 5 numaralı toparlama kullanın ve diğer toparlamalarla birlikte karnı torakstan çekin. Testisleri ve bitişik dokuları sinek karkası15'ten hızla izole edin ve ringer tamponunun yeni bir damlasına aynı slaytta yerleştirin.</li>
	<li>Tüm testisler hazır olduğunda, testisleri kalan dokulardan (aksesuar bezi ve dış cinsel organ) temizleyin.</li>
	<li>Parçalanmış testisleri içeren damladan fazla tamponu çıkarın. Tipik olarak, 200 μL ucun üzerine monte edilmiş 10 μL uçlu bir p200 pipet kullanın. Bu, sıvı fazlalığının küçük bir diyafram açıklığından çıkarılmasını sağlar. Parçalanmış testisleri içeren damlanın üzerine% 4 paraformaldehit fiksatif bir damla yerleştirin ve ardından testisleri hızlı bir şekilde taze fiksatif çözelti içeren bir tüpe aktarın.</li>
</ol>2. Paraformaldehit fiksasyonu
<ol><li>Kullanımdan hemen önce 2 mL fiksatif çözelti hazırlayın: 2 mL silikonlu mikrosenj tüpte 30 μL NP40% 20 artı 1200 μL hepatik ile fosfat tamponlu salin (PBS) ile seyreltilmiş% 4 paraformaldehitin 600 μL'si. 600 μL aliquots olarak hazırlanan ve -20 °C'de depolanan PBS'de %4 paraformaldehit kullanın. Dikkat: Heptan ve paraformaldehit toksiktir ve duman kaputunda ele alınmalıdır. Ev sahibi enstitünün yönetmeliklerine göre bunları atın.</li>
	<li>Testleri geniş diyafram ucuna sahip bir p200 mikropipette kullanarak PFA/NP40/heptane çözeltisine aktarın. Tüpü elle 30 sn boyunca yukarı ve aşağı sallayın. Testisleri oda sıcaklığında 30 dakika boyunca döner karıştırıcıya sabitleye devam edin.</li>
	<li>Döner karıştırıcıyı durdurun ve faz ayrımına izin vermek ve testislerin tüpün dibine yerleşmesini sağlamak için tüpü bir tüp rafı içine yerleştirin.</li>
	<li>Tüm heptane ve fiksatif çözeltiyi çıkarın ve testisleri 1x PBS ile üç kez hızlı bir şekilde durulayın.</li>
	<li>Temiz bir 200 μL bireysel tüpe aktarın. Kullanılan antikor miktarlarını en aza indirmek için bu küçük tüplerdeki tüm inkübasyonları gerçekleştirin.</li>
	<li>Hem P200 hem de P10 ucu takılı 200 μL mikropipette kullanarak fazla tamponu çıkarın. Testisleri aspire etmemeye dikkat edin.</li>
	<li>Tüm numuneler hazır olana kadar testisleri buz üzerinde 1x PBS'de tutun.</li>
</ol>3. Çözeltide antikor lekesi
<ol><li>PBS'yi atın ve hücre zarlarını permeabilize etmek için oda sıcaklığında 30 dakika boyunca testisleri% 0,3 PBS-T'de bırakın.</li>
	<li>Aynı tamponda önceden inkübasyon artı 1 saat boyunca% 5 normal keçi serumu (NGS).</li>
	<li>%0,3 PBS-T artı %5 NGS(Malzeme Tablosu)ile seyreltilmiş birincil antikorun 200 μL'sini ekleyin.</li>
	<li>Birincil antikoru bir gecede 4 °C'de (veya oda sıcaklığında) bir rocker üzerinde hafif ajitasyonla kuluçkaya yatırın.</li>
	<li>Oda sıcaklığında bir rocker üzerinde 15 dakika boyunca% 0.3 PBS-T'de 3 kez yıkayın. Testislerin yerçekimi ile tüpün dibine yerleşmesine izin verin.</li>
	<li>DyLight konjuge serisinden 1:500 seyreltilmiş 200 μL ikincil antikor ekleyin.</li>
	<li>İkincil antikorun oda sıcaklığında 4 saat boyunca karanlık bir odada kuluçkaya yatırın.</li>
	<li>Oda sıcaklığında bir rocker üzerinde 15 dakika boyunca% 0.3 PBS-T'de yıkayın. Daha sonra her seferinde oda sıcaklığında bir rocker üzerinde 30 dakika boyunca% 0.2 PBS-T ve% 0.1 PBS-T ile tekrarlayın. Deterjanı çıkarmak için 30 dakika boyunca 1x PBS'de son bir yıkama yapın.</li>
	<li>15 dakika boyunca 1x PBS'de 1 μg/mL'de 180 μL DAPI çözeltisi ekleyin. Stok çözeltisi H2O'da 10 mg/mL'dir. PBS'de stok çözeltisi yapmaktan kaçının, bu da daha büyük spermatositlerde dağınık kromatin üzerindeki DAPI boyama sinyalini azaltabilir.</li>
	<li>Her biri 5 dakika boyunca 3x yıkayın.</li>
	<li>Temiz bir slayt üzerinde bir daire çizmek için hidrofobik bir bariyer kalemi kullanın.</li>
	<li>Testisleri aspire edin ve slayda koyun. Geniş diyafram ucunun %0,1 PBS-T ile önceden durulanmasını test etmenin uyrmaya yapışmasını önler. Fazla PBS'yi kaldırın.</li>
	<li>Citifluor AF1'den bir damla (yaklaşık 100 μL) ekleyin.</li>
	<li>Hava kabarcıklarını hapsetmekten kaçınmaya dikkat ederek, dokunun üzerine hafifçe bir cam kapak yerleştirin.</li>
	<li>Kapak kapağını şeffaf oje kullanarak kaydırağa kapatın.</li>
	<li>Slaytları mikroskopta gözlemleyin.</li>
</ol>4. Kolokalizasyon analizleri
<ol><li>Konfokal görüntüler elde edin. Z çözünürlüğünde 0,5 veya 1 μm çözünürlüğünde zeiss LSM 710 konfokal mikroskop (63x Plan Apokromatik yağ) kullandık.</li>
	<li>Bağımsız testislerden farklı hücreler seçin.</li>
	<li>Görüntüleri işleme yazılımına (örneğin, imaris) içe aktarın.</li>
	<li>Komşu çekirdeği dışlamak için çekirdeği manuel segmentasyonla tanımlayın. Coloc yazılımını kullanın; Pearson'ın korelasyon katsayılarını (PCC' ler) 2 florofor arasındaki tüm hacimde toplar.</li>
</ol>

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Yazarların açıklayacak bir şeyi yok.

Bu protokol, tüm mount yetişkin Drosophila testislerinin başarılı bir şekilde immünoblabeling için bir yöntem sağlar. Diğer prosedürlerde bildirildiği gibi, spermatositleri engelleme riski taşıyan spermin varlığını en aza indirmek için genç erkeklerin kullanılması gerekir (hatta eklosyondan sonra yaşı 20 saatten daha az bir süreye kısalttık). Diseksiyon hızlı olmalı ve antikor seyreltmesi sinyal-gürültü oranını optimize etmek için ayarlanmalıdır21,...

Drosophila testisleri nükleer mimarinin incelenmesi için değerli bir modeldir. Mitotik spermatogonyal hücrelerde, nükleer hacim büyük ölçüde kromatin tarafından işgal edilir. Bu hücreler dört tur bölünmeye uğrar ve 16 birincil spermatosit kisti üretir. Sonraki birkaç gün boyunca, spermatositler 6 gelişim aşamasından (S1-S6) geçer, hacim olarak büyük ölçüde artar, yaklaşık olarak 20 kat1,2. Nükleer morfolojilerini de değiştiriyorlar. Lamin Dm0 tarafından ortaya çıkan çekirdeğin ana hatları düzensiz hale gelir ve derin invaginasyonları gösterir1,3. Buna kromatin organizasyonu 1 , 4 ,5,6,7 'de kapsamlı gen ekspresyasyonu değişiklikleri ve modifikasyonları eşlikeder. Ara evre kromozomları, S4-S5 evresinde iyi tanımlanmıştır ve 2 büyük çift değerli otomoz ve çekirdekle sabitlenmiş X-Y çiftine karşılık gelen üç ayrı kütle oluşturur.
Mad1 başlangıçta mitotik kontrol noktasının önemli bir bileşeni olarak izole edildi(8'degözden geçirildi). Interfazda, öncelikle nükleer zarfta değil, aynı zamanda nükleoplazma9, 10,11'dede bulunur. Drosophila testislerinde, Mad1'in nükleoplazmada belirgin olduğunu, iki büyük otozomal kromatin kütlesi ile yakından ilişkili olduğunu ve XY kromatin ile daha az ölçüde ilişkili olduğunu bildirdik. Kromatinle ilişkili bu yapıyı MINT (Mad1 içeren IntraNükleer Bölge)12olarak tanımladık. Diğer dört protein spermatositlerdeki MINT'lerle ilişkiliydi: nükleoporin Mtor / Tpr, SUMO peptidaz Ulp1, mitotik kontrol noktası proteini Mad2 ve Polikomla benzeri bir kompleksin alt bireyi Raf212.
MINT'lerin tanımını tamamlamak için antikor kullanmamız gerekiyordu ve testislerde genellikle immünostaining ile karşılaşılan teknik sorunlarla karşı karşıya kaldık: testislerin derinliğinde, özellikle de büyük spermatositlerde önemli bir homojenlik ile sonuçlanan zayıf bir geçirgenlik. Squashed preparatları antikor erişimini artırdı, ancak sıkıştırılmış görüntülere yol açtı, 3D analizleri kısıtladı ve kolokalizasyon da dahil olmak üzerenicel floresan ölçümünü önledi. Antikor penetrasyonu sorunu da % 0.3 Na deoksikollat13gibi permeabilizasyon edici ajanlar tarafından ele alınabiliyordu, ancak bu prosedür elimizde tekrarlanabilir sonuçlar vermedi. Birçok ortak etiketleme deneyi yaptığımız için, permeabilizasyon protokolünü geliştirmeye baktık. % 1 NP40 artı heptan kombinasyonu, özellikle büyük spermatositlerin incelenmesinde daha fazla tekrarlanabilirlik ile sonuçlandı.
Bu protokol, süspansiyondaki testislerin sabitlenmesi ve immünostainingini gerçekleştirir, numune kalitesini artırır, tekrarlanabilirliği artırır ve konfokal mikroskopi için uygun hale getirir. Protokolü, bazı nükleer zarf proteinlerinin büyük spermatositlerin nükleoplazmik yapılarıyla olan mekansal ilişkilerini daha iyi tanımlamak için kullandık. Bu yöntem, spermatosit gelişimine eşlik eden değişikliklerin, örneğin kromatin, nükleoplazma ve nükleer gözenekler de dahil olmak üzere çekirdeğin dinamik yapısal değişikliklerinin daha iyi araştırılmasına izin verecektir.

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			<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>
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			<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

Drosophila testisleri kök hücre biyolojisi, nükleer mimari, meiosis ve sperm gelişimi dahil olmak üzere biyolojik süreçleri incelemek için güçlü bir model sistemidir. Bununla birlikte, tüm Drosophila testislerinin immünolabelingi genellikle antikor penetrasyonu nedeniyle lekelemenin önemli ölçüde homojen olmayanlığı ile ilişkilidir. Ezilmiş preparatlar, analizlerin 3D kalitesini azalttığı için sorunun sadece kısmen üstesinden gelmektedir. Burada, sıvı ortamda immünolabeling ile birlikte fiksasyon sırasında NP40 ve heptane kullanan tam montajlı bir protokol açıklıyoruz. Konfokal mikroskopiye uygun hacmi tekrarlanabilir ve güvenilir etiketleme ile birlikte korur. Konfokal bölümlerden spermatosit çekirdeğinin 3D rekonsesinin farklı örneklerini gösteriyoruz. İntra-ve inter-testes tekrarlanabilirliği, farklı genotiplerden tek hücreler arasındaki floresanların 3D nicelleştirilmesine ve karşılaştırılmasını sağlar. Bu protokolü ve testislerin en büyük hücreleri olan S4-S5 spermatositleri üzerindeki uygulamalarını göstermek için intranükleer MINT yapısının (Mad1 içeren Intra Nükleer Bölge) farklı bileşenlerinin yanı sıra nükleer gözenek kompleksi ile ilişkili iki bileşen kullandık.

Drosophila testislerinin yeterli lekelenmesine yönelik en büyük engel, ezilmiş preparatlar kullanılarak kısmen çözülmüş olan ancak 3D analizlerin (örneğin 3D temsili veya kolokalizasyon ölçüsü) kısıtlanması pahasına antikorların sınırlı penetrability'dir. Prosedür tek tip etiketlemeye izin veriyor ve testislerin tüm hücrelerinin hacmini koruyor. Burada yöntemin illüstrasyonunu S4-S5 spermatositlerinin 3D temsiline odakladık, çünkü en büyük hücrelerdir ve böylece ezilme deformasyonuna...

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

Burada, konfokal mikroskopi için uygun ve aynı zamanda tekrarlanabilir ve güvenilir etiketlemeye izin vermek için Drosophila testislerinin tüm montaj hazırlığı ve immünostainingi için geliştirilmiş bir protokol sunuyoruz. Bu protokolü, büyük S4-S5 spermatositleri üzerindeki kolokalizasyon deneylerinin 3D gösterimi ve niceliği ile göstermektedir.

Büyük Spermatositlerin 3D Konfokal Analizi için Tüm Kazdağları Drosophila Testlerinin İmmünostaining

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 Görüntüleme.  Université de Paris, F 75006, Paris, France. Burada, konfokal mikroskopi için uygun ve aynı zamanda tekrarlanabilir ve güvenilir etiketlemeye izin vermek için Drosophila testislerinin tüm montaj hazırlığı ve immünostainingi için geliştirilmiş bir protokol sunuyoruz. Bu protokolü, büyük S4-S5 spermatositleri üzerindeki kolokalizasyon deneylerinin 3D gösterimi ve niceliği ile göstermektedir.

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.

Erken Civciv Embriyolar in situ Hibridizasyon Çift Tüm Dağı

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

Biyoloji

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.

Chick Tüm Dağı Antikor Boyama Yöntemi

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.

Zebra balığı Tüm Dağı Yüksek Çözünürlüklü Çift Floresan Yerinde Hibridizasyon

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.

Tüm Maymun Beyin Büyük Ölçekli Protein Algılama için Toplu immün

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

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Embriyonik Ekstremite Cilt damarları Tüm montaj İmmünohistokimyasal Analizi: Embriyo vasküler dallanma Morphogenesis Eğitim Model Sistemi

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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.

İzolasyonu Drosophila melanogaster Testisler

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.

Tüm Dağı<em&gt; In situ</emE11.5 fare embriyolarına E8.5 ve&gt; Hibridizasyon

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.

Tüm Dağı RNA Floresan<em&gt; In situ</emVe&gt; Hibridizasyon<em&gt; Drosophila</em&gt; Embriyolar

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;

Spermatogenez sitolojik analizi: Canlı ve Sabit Hazırlıklar<em&gt; Drosophila</em&gt; Testisler

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.

Aktif Kaspaz 3 Algılama Tüm montaj immünofloresans tarafından Zebra balığı Embriyolar Apaptozun Analizi

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