Bu çalışma ABD Ulusal Sağlık Enstitüleri tarafından desteklenen HL082798 ve HL111580 verir.

1.. Böbrek Doku Bölümler <ol><li> Formalin-sabit (% 10) parafin-gömülü böbrek bölümleri slaytlar analizden önce birkaç ay boyunca saklanabilir bir Microm HM 355 S. kullanılarak 5 mm kalınlığında mikroskop lamı üzerine kesilir. </li></ol> 2. Çözüm Hazırlığı <ol><li> Bir Otoklavlanabilir vida üst şişede GKD 2 O 1x PBS 1 L hazırlayın. GKD 2 O. 1 L ile bir şişe doldurun Her şişe, kapak DEPC 1 ml ekleyin ve şiddetle çalkalanır. Çözümler RNaz&#39;larına yok etmek ve daha sonra otoklavlanmasıdır gece oda sıcaklığında bekletin. PBS-T için 400 ml Tween-20 1x PBS L 1 ekle. </li><li> Proteinaz K tamponu (50 mM Tris-HCl, pH 8.0 ve DEPC 1 mM CaCl2 su muamele edilmiş) hazırlayın. </li><li> PBS-T içinde% 0.2 glisin içeren bir çözelti hazırlayın. </li></ol> 3. Doku Hazırlanması <ol><li> Hibridizasyon tamponu büyük bir hacim% 50 Formamid oluşan (50-100 ml), 5x SSC,% 0.1 Tween, 9,2 hazırlanmasıpH 6, 50 ug / ml heparin ve DEPC içinde 500 ug / ml maya RNA için ayarlama için mM sitrik asit GKD 2 O. muamele -80 ° C&#39;de 1 ml&#39;lik numuneler hazırlayın ve depoya Böl </li><li> Her biri 5 dakika için taze ksilen içinde 3 kez yıkayarak dokudan parafin çıkarın. </li><li> GKD 2 O. etanol konsantrasyonunu (% 100,% 75,% 50 ve% 25) azaltmada 5 dakika inkubasyon ile doku bölümleri rehidrate </li><li> Tam olarak 1 dakika boyunca doku bölümleri (yaklaşık olarak 0.4-0.5 mi) karşılamak için yeterli DEPC ile slaytlar tedavi. Emin olun dokular kuru değil. </li><li> DEPC yılında slaytlar uygun bertarafı için DEFPC içeren atık toplama, 5 dakika boyunca iki kez PBS-T tedavi durulayın. Tüm reaktifler DEPC bu noktadan RNaz&#39;larına ortadan kaldırmak için tedavi edilmelidir. </li><li> Ön ısıtılması Proteinaz K tamponu ve 10 ug / ml &#39;lik bir nihai konsantrasyona kadar proteinaz K ilave edin. Proteinaz K solüsyonu ile doku bölümleri kapak ve 5 dakika boyunca 37 ° C&#39;de inkübe edilir. </li><li> Çabuk proteinas çıkarıne K çözeltisi ve 30 saniye için PBS-T içinde% 0.2 glisin ekleyin. </li><li> DEPC içinde durulayın slaytlar 30 saniye, her iki kez PBS-T işlemden geçirildi. </li><li> 10 dakika boyunca taze% 4 paraformaldehid içinde bölümleri düzeltildi. </li></ol> 4. Melezleme <ol><li> Hibridizasyon tamponu 50 ul (% 50 formamid, 5x SSC,% 0.1 Tween, pH 6, 50 ug / ml heparin, 500 ug / ml maya RNA için ayarlama için 9.2 mM sitrik asit) doku kesiti başına ekleyin ve RNAse serbest HybriSlips ile kapağı doku bölümleri biraz daha büyük kesilir. </li><li> 3 &#39;digoksigenin işaretli prob (sonda genellikle Tm için belirlenmiş DNA melezleştirme sıcaklığında 2 saat boyunca nem kontrollü bir hibridizasyon fırınında Prehybridize bölümler -21 ° C, miR-382 için (örneğin, 54 ° C, DNA Tm = 75 ° C, sonda ), bölmeyi tutmak için% 50 formamide/50% 5x SSC batırılmış doku laboratuar mendil ile nemlendirilir. </li><li> MiRNA probu, pozitif kontrol (U6 küçük nükleer RNA) ve negatif kontrol (şifreli sulandırmakhibridizasyon tamponu içinde 40 nM&#39;ye sekans) (tampon maddesi 75 ul), her bölüm gereklidir. </li><li> 5 dakika boyunca 65 ° C&#39;de hibridizasyon tamponu içinde probu ısıtın. </li><li> Melezleme fırını slaytları çıkarın ve melezleme gelen lamelleri ve aşırı hibridizasyon tamponu çıkarın. </li><li> Doğrudan dokuya, doku Bölüm başına prob ihtiva eden bir tampon içinde 75 ul ekle. Doku bölümleri kapsayacak kadar sadece büyük HybriSlips ile doku örtün. </li><li> Slayt tutucu düzeyde tutulması, Aşama 4,2 (yaklaşık 16 saat) arası bir sıcaklıkta gece boyunca inkübasyon için melezleme fırını dönün. </li></ol> 5. Sertliği Yıkama <ol><li> Dokudan lamel serbest bırakmak için sadece lamelleri bir kenarının altında hibridizasyon sıcaklığa ısıtılmış 200 ul 2x SSC ekleyin. Slayt eğerek lamel çıkarın. </li><li> 30 dakika her biri için melezleştirme sıcaklık 3x% 50 formamid,% 50 2 x SSC içinde 200 ul slaytlar yıkayın. </li><li> DurulamaSlaytlar düşük hızda orbital çalkalayıcı üzerinde 5 dakika her biri için oda sıcaklığında PBS-T içinde 5 kat. </li></ol> 6. Bulma <ol><li> Düşük hızda orbital bir karıştırıcı üzerinde oda sıcaklığında 1 saat süreyle (% 2 keçi ya da at serumu, 2 mg / ml PBS-T içinde BSA) bloke slayt inkübe edin. </li><li> 1:100 bloke edici tampon içinde bir anti-DIP-AP Fab fragmanları seyreltin. Doku Bölüm başına 100 ul ekleyin ve HybriSlips ile kapak bölümünü kaplayacak kadar sadece büyük kesti. </li><li> 4 ° C&#39;de, gece boyunca (yaklaşık 16 saat), bir nemlendirilmiş oda içinde antikor inkübe edin. </li><li> Düşük hızda orbital çalkalayıcı üzerinde 5 dakika her biri için, PBS-T 7x slaytlar yıkayın. </li><li> 5 dakika her biri için, 3x (100 mM TrisHCI pH 9.5, 50 mM MgCl2, 100 mM NaCl,% 0.1 Tween 20) ön-arka tampon maddesi içinde slaytlar yıkayın. </li><li> AP tamponuna NBT / BCIP solüsyonu (200 mi μl/10 tamponu) </li><li> Tamamen tüm doku bölümleri kapsayacak şekilde her slayt için seyreltilmiş NBT / BCIP çözeltisi 400-500 ml ekleyin. Karanlık, seviye, gösterilmeyen nem Develop4-5 saat için IED odası. </li></ol> 7. Montaj Slaytlar <ol><li> 5 dakika her biri için etanol konsantrasyonlarını (% 25,% 50,% 75,% 100) artan bölümleri kurutmak. </li><li> Bölümleri temizlemek için ksilen 5x inkübe. </li><li> Dağı Permount montaj orta ve kuru gecede slaytlar. </li></ol>

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Ifşa hiçbir şey yok.

Bu makalenin amacı formalin sabit böbrek dokularında iyi çalışır in situ hibridizasyon miRNA için bir protokol açıklar oldu. Bu protokol üzerinde çalışırken boyama dışlayıcı birçok önemli kaynakları tespit edilmiştir. Bu noktalara dikkat ve özen boyama objeyi önlemek ve başarılı ISH çalıştırmak olasılığını artırabilir. Haline doku bölümleri uzun inkubasyon sırasında kuruduğunda boyama dışlayıcı en önlenebilir nedenlerinden biri oluşab...

MicroRNA endojen olarak üretilir kodlayıcı olmayan RNA&#39;lar (yaklaşık 22 nükleotid uzunluğunda) küçüktür. Bunlar genellikle translasyon baskı veya mRNA degradasyon yoluyla protein ekspresyonunu engellemek için çalışır. eksik tamamlayıcılık mRNA hedeflere miRNA&#39;ların bağlamak mümkün, tek bir miRNA çoklu hedefleri bastırmak için yapım. Hücre tipleri ve yapılarının miRNA&#39;lar ifade anlayış mekanizmaların anlaşılmasında önemli bir parçası olan aracılığıyla miRNA ifade etkisi hücre ve doku fenotipleri değişiklikler. Bu tür sıralama MiRNA, QPCR ve Kuzey beneklemesi gibi yöntemler bütün dokularda miRNAs tespiti için kullanılabilecek olmakla birlikte, bu yaklaşım, bir de, belirli bir doku içinde gelen hangi belirli bir hücre tipi belirlemek için izin vermez. Bu yöntemlerle analizden önce hücresel ve yapısal bileşenlerin Diseksiyon çok zor olabilir ve yeterli izolasyonların elde etmek için gerekli olan koşullar al yol açabilirgen ekspresyonu ya da RNA&#39;lar parçalanmasında önemli ola. in situ hibridizasyon microRNA dokularda microRNA konumu ve ekspresyon seviyelerini görselleştirmek için kullanılan bir yöntemdir. Bu teknik, böbrek gibi heterojen yapıların, oluşan dokularda, özellikle değerlidir. MicroRNA böbrek gelişimi 2,3 ve fizyoloji 4 düzenleyici bir rol oynadığı gösterilmiştir. microRNA sentezleme değişiklikler, aynı zamanda, 5-10 fibrosis, diabetik nefropati 7, renal karsinom 11,12 ve akut böbrek hasarı 13 gibi renal patoloji ile ilgili olduğu gösterilmiştir. Bizim araştırmada, böbrek dokularında in situ hibridizasyon mıkroRNA optimize sağlık ve hastalık 14 hem de miRNA ifade tesisinin yapısal yeri saptanması için değerli olduğunu bulduk. Farklı microRNA boru şeklindeki ve hücresel ifade belirlenmesi önemlidir, çünkü ta onların düzenlenmesirgets hücre fonksiyonları bağlı olabilir. Hastalıklı eyaletlerde bu miRNA ifadesi değişiklikler fonksiyonunu etkileyen olabilir belirlemek için de önemlidir. Burada tarif edilen yöntemin amacı Kloosterman et al. 15, diğer araştırmacılar 16,17 tarafından geliştirilen mevcut ISH yöntemleri üzerine inşa etmek için, ve bu Exiqon 1 tarafından verilir ve formalin sabit böbrek dokularında yöntemini optimize eder. Biz başarıyla tek taraflı üreter 18 ile böbrek mıkroRNA miR-382 ifadesinde belirgin bölgesel farklılıkları belirlemek için bu yöntemi kullandık. Bu yaklaşım ek optimizasyonu ile, hem de diğer dokuları ile kullanılabilir.

<table border="0" cellpadding="0" cellspacing="0" width="481">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P6148</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">PBS</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:137px;">70011044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Diethyl Pyrocarbonate</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">D5758</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Tween-20</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">P1379</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Xylenes</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">534056</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Ethanol</td>
			<td style="width:71px;">Ultra Pure</td>
			<td style="width:137px;">200CSPTP</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Tris-HCl</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15506-017</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">CaCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2536</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Glycine</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4059-00</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Citric Acid</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">C2404</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Formamide</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">F9037</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">20x SSC Buffer</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">15557-044</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Heparin</td>
			<td style="width:71px;">SAGENT</td>
			<td style="width:137px;">25021-400-30</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Yeast RNA</td>
			<td style="width:71px;">Life Technologies</td>
			<td style="width:137px;">AM7118</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">MgCl2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:137px;">M8266-1004</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">NaCl</td>
			<td style="width:71px;">J.T. Baker</td>
			<td style="width:137px;">4058-01</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Proteinase K</td>
			<td style="width:71px;">Fermentas</td>
			<td style="width:137px;">EO0491</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- miRNA probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">miR dependent-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">3&#8217; DIG- Scrambled miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99004-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">3&#8217; DIG- U6 miR probe</td>
			<td style="width:71px;">Exiqon</td>
			<td style="width:137px;">99002-05</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Anti-Digoxigenin-Ab Fab</td>
			<td rowspan="2" style="width:71px;">Roche</td>
			<td rowspan="2" style="width:137px;">11093274910</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Fragments</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">NBT/BCIP</td>
			<td style="width:71px;">Roche</td>
			<td style="width:137px;">11681451001</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Permount</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">SP15-500</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Coverslips</td>
			<td style="width:71px;">Fisher</td>
			<td style="width:137px;">Fit to tissue size</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Microtom HM 355 S</td>
			<td style="width:71px;">Thermo Scientific</td>
			<td style="width:137px;">23-900-672</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">In-slide Out Hybridization Oven</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">241000</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Aluminum Tray Assembly</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403973</td>
			<td style="width:79px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:195px;">Stainless Steel Rack Insert</td>
			<td style="width:71px;">Boekel</td>
			<td style="width:137px;">C2403754</td>
			<td style="width:79px;"></td>
		</tr>
	</tbody>
</table>

microrna in situ hybridization for formalin fixed kidney tissues

Bu yazıda microRNA problar etiketlenmiş digoksijenin ile in situ melezleme yoluyla böbrekte miRNA kolorimetrik tespiti için bir yöntem tarif eder. Başlangıçta Kloosterman ve Exiqon miRNA sondalar 1 ile geniş kullanım için meslektaşları tarafından geliştirilen bu protokol, böbrek dokularında miRNA analizinde doğal zorlukları aşmak için modifiye edilmiştir. Bunlar yapı tespiti ve kalıntı prob ve antikor çıkarmak zor gibi konuları içerir. Nispeten ince kullanımı olup, 5 mm kalınlığında, güçlü bir prob sinyal hücrelerinde muhafaza ederken, böbrek yapıları net bir görünüm için izin verilen doku kesitleri. Ayrıca, prob konsantrasyonu ve inkübasyon koşulları Düşük arka plan ve spesifik olmayan sinyal ile mıkroRNA ifade görselleştirme kolaylaştırmak için optimize edildi. Burada, optimize edilmiş prosedür protokolü sonunda slaytların montajı yoluyla ilk doku toplanması ve hazırlama kaplama açıklanmaktadır. Temel componeBu protokolün nts diğer doku ve hücre kültürü modelleri uygulama için değiştirilebilir.

Hibridizasyonlar, inkubasyon sırasında kurutulmuş veya NBT / BCIP gelişimi sırasında daha koyu lekelere sona genellikle yıkama aşamaları olur bir doku kesitinin alanları. 1 HybriSlip kenarının kaymış olduğu bir böbrek bölümünün bir bölümünü göstermektedir, Şekil kısmen susuz olmak sağlayan doku. Kalan adımda rehidrasyon ve içerisinde rağmen, susuz kısmında sinyal yapay yüksektir. NBT-BCIP gelişme zamanını kontrol önemi, ...

Watch this Scientific Journal Video about MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues at JoVE.com

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

Bu makalede, formalin içerisinde tespit edilmiş böbrek bölümlerinde microRNA ifade Renkölçer tespiti için optimize edilmiş bir in situ hibridizasyon protokolü tarif eder.

MicroRNA<em&gt; In situ</em&gt; Formalin Sabit Böbrek Dokular Hibridizasyon

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

microrna-in-situ-hybridization-for-formalin-fixed-kidney-tissues

Research

JoVE Journal

Biology

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

14.8K Görüntüleme.  Medical College of Wisconsin.  Bu makalede, formalin içerisinde tespit edilmiş böbrek bölümlerinde microRNA ifade Renkölçer tespiti için optimize edilmiş bir in situ hibridizasyon protokolü tarif eder. 

Video: MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

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

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 ...

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Floresan In situ İnsan Sperm hibridizasyon (FISH) Protokolü

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In Situ Hybridization for the Precise Localization of Transcripts in Plants

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene expression. Microarray and next generation sequencing approaches are being used to investigate developmental, physiological and stress response processes, dissect epigenetic and small RNA pathways, and build large gene regulatory networks1-3. While these techniques facilitate the simultaneous analysis of large gene sets, they typically provide a very limited spatiotemporal resolution of gene expression changes. This limitation can be partially overcome by using either profiling method in conjunction with lasermicrodissection or fluorescence-activated cell sorting4-7. However, to fully understand the biological role of a gene, knowledge of its spatiotemporal pattern of expression at a cellular resolution is essential. Particularly, when studying development or the effects of environmental stimuli and mutants can the detailed analysis of a gene's expression pattern become essential. For instance, subtle quantitative differences in the expression levels of key regulatory genes can lead to dramatic phenotypes when associated with the loss or gain of expression in specific cell types. 
Several methods are routinely used for the detailed examination of gene expression patterns. One is through analysis of transgenic reporter lines. Such analysis can, however, become time-consuming when analyzing multiple genes or working in plants recalcitrant to transformation. Moreover, an independent validation to ensure that the transgene expression pattern mimics that of the endogenous gene is typically required. Immunohistochemical protein localization or mRNA in situ hybridization present relatively fast alternatives for the direct visualization of gene expression within cells and tissues. The latter has the distinct advantage that it can be readily used on any gene of interest. In situ hybridization allows detection of target mRNAs in cells by hybridization with a labeled anti-sense RNA probe obtained by in vitro transcription of the gene of interest. 
Here we outline a protocol for the in situ localization of gene expression in plants that is highly sensitivity and specific. It is optimized for use with paraformaldehyde fixed, paraffin-embedded sections, which give excellent preservation of histology, and DIG-labeled probes that are visualized by immuno-detection and alkaline-phosphatase colorimetric reaction. This protocol has been successfully applied to a number of tissues from a wide range of plant species, and can be used to analyze expression of mRNAs as well as small RNAs8-14.

In Situ Hybridization for the Precise Localization of Transcripts in Plants

The in situ hybridization protocol described here allows a direct localization of mRNA and small RNA expression at the cellular level with high sensitivity and specificity. The procedure is optimized for paraffin-embedded plant tissue sections, is applicable to a wide range of plants and tissues, and can be completed within ten days.

Bitkilerde Transkriptler Hassas Yerelleştirme Situ Hibridizasyon

With the advances in genomics research of the past decade, plant biology has seen numerous studies presenting large-scale quantitative analyses of gene ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Radyoaktif In situ</emDoku Çeşitli Gen Patterns Saptayan> Hibridizasyon

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Floresans tarafından viral RNA tespit edilmesi<em&gt; In situ</em&gt; Hibridizasyon (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNA Floresan tarafından Tek hücreli Gen Transkripsiyon Analizi<em&gt; In Situ</em&gt; Hibridizasyon (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Floresan ile birlikte Kromozom çoğaltılıyor Zamanlama<em&gt; Yerinde</em&gt; Hibridizasyon

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Formalin gelen Proteomic Numune Hazırlama parafin blok doku Sabit ve

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...