We thank the Advanced Microscopy Unit (School of Life Sciences, University of Nottingham) and the Histology team of MRC Human Reproductive Sciences Unit (Edinburgh), the Institute of Neuroscience at the ULB and the FNRS for help and support. This work was supported by MRC (MR/L001047/1 to A.D.J.).

Tüm hayvan-ilgili prosedürler Üniversitesi&#39;nin Nottingham etik inceleme kurulu ile uygun olarak yapıldı. 1. immün için uygundur Doku Hazırlama seçilmesi <ol><li> Daha önce tarif edildiği gibi, 25 vahşi tip CD1 fare embriyoları ve yetişkin beyin dokularının parafine gömülü bölümü oluşturur. % 4 formaldehit (FA) veya yöntemi 25 immün süreyle% 4&#39;lük paraformaldehit (PFA) ile ya tespit edilmiş olan beyin doku bölümleri kullanın. NOT: parafin doku kesitlerinin kullanılması antikor etiketleme önce de-ağda gerektirir. 2 ile tedavi, -, DNA&#39;nın denatüre edilmesi için protokol kullanılır 4 M hidroklorik asit (HCI) çoğu antijen alma stratejileri ile uyumlu değildir, Protein ile 5MC oksidasyon türevlerinin eş saptanması için kriyo-mikrotom veya bir kısmından kullanılmasını tavsiye belirteçler. </li></ol> 2. Mum Parafin Gömülü Doku Bölümler &lt;<html> / P&gt; <ol><li> Çalışan bir sınıf II güvenlik kabininde, ksilen, oda sıcaklığında 10 dakika için 2 defa her biri ile dolu bir Coplin kavanoz parafine gömülü doku kesitleri yıkayın. NOT: Eksik parafin giderme tutarsız boyanma yol açabilir gibi taze ksilen kullanmak önemlidir. </li><li> de-ağda sonra hızlı bir şekilde art arda 95, 75 yıkanarak doku bölümleri rehidrate, ve oda sıcaklığında 10 dakika her biri% 50 etanol. </li></ol> 3. Sabitleme ve kriyo ve Mikroskobik Bölümler Permeabilization <ol><li> Oda sıcaklığında 15 dakika boyunca buz gibi soğuk% 4 PFA ya da 4% F ya yerleştirilerek rehidre doku bölümleri düzeltildi. </li><li> Oda sıcaklığında 5 dakika boyunca PBS içinde bölümler (Fosfat tampon salin) yıkanarak fazla Fiksatif çıkarın. </li><li> Oda sıcaklığında 30 dakika boyunca (PBS içinde% 0.5 Triton X-100) PBX ile doldurulmuş bir Coplin kavanoza yerleştirilmesiyle doku bölümleri geçirgenliği. PBT (PBS içerisinde% 0.01 Tween 20) ile kısa bir süre bölümleri yıkanarak fazla PBX çıkarın. </li></ol>Oxi-5MC Türevleri jove_title &quot;&gt; 4. Immün <ol><li> DNA depürinasyon oda sıcaklığında 60 dakika boyunca 2 N HCI içinde geçirgen kesitleri yerleştirin. NOT: Her ne kadar HCl daha yüksek konsantrasyonlarda (örneğin, 4 H) talebi daha verimli, DNA&#39;nın denatüre edilmesi için, bunlar DAPI ile oksi-MCS eş saptanması için uyumlu değildir. </li><li> HCl&#39;yi nötralize etmek için oda sıcaklığında 30 dakika boyunca 10 mM Tris-HCl (pH 8.5) bölümleri yerleştirin. Alternatif olarak, 5 dakika PBS içinde her seksiyonu üç kez yıkayın. Oda sıcaklığında 5 dakika boyunca PBT bölümleri inkübe edin. </li><li> Dikkatle hafifçe slayt sallayarak herhangi bir aşamada tamamen kurumasını doku bölümü izin vermeden doku bölümü çevredeki sıvıyı çıkarın. bölüm dokunmadan bölümü kuşatmak için hidrofobik bir bariyer kalem kullanın. NOT: hidrofobik bariyer kalem doku leke ve birden çok bölüm ayırıcı ile boyanmış izin verebilirsiniz gerekli antikor hacmini azaltırAynı slaytta t antikorlar. </li><li> nemli bir oda içinde oda sıcaklığında 1 saat için bloke etme çözeltisi (PBS içinde% 10 sığır serumu albümini) 100 ul bölümleri inkübe edin. NOT: Bu adımı atlama modifiye sitozin bazları boyama verimini etkilemez olmaz. </li><li> Fare monoklonal anti-5hmC 5,000 seyreltme ve 1: nemli bir oda içinde oda sıcaklığında 1 saat için bloke etme çözeltisi tavşan poliklonal anti-5caC birincil antikor 1000 seyreltme 1 100 ul doku bölümleri inkübe edin. Gerekirse Alternatif olarak, 4 ° C&#39;de bir gece inkübasyona gerçekleştirin. NOT: Primer antikor olmadan işlenen Bölümler boyama işlemi için uygun olarak negatif kontroller hizmet edebilir. 12.5 gün pozitif kontrol olarak kullanılabilir 5caC, 5FC ve 5hmC 20 zenginleştirilmiş coitum murin embriyonik beyin kesitlere. </li><li> Bir Cop içinde 5 dakika boyunca PBT ile dolu bir Coplin kavanoza her üç kez bölümleri yıkanarak fazla antikorlarıRT&#39;de lin kavanoz. NOT: yıkama solüsyonlarının hacmini arttırmak arka plan boyama azaltabilir. </li><li> Fazla PBT çıkarın ve gerekirse PBT hidrofobik bariyer zayıflatabilir deterjan ihtiva gibi hidrofobik bariyer kalem ile tekrar bölümleri kuşatmak. </li><li> Keçi anti-tavşan HRP ile konjüge edilmiş antikor 400 seyreltme ve 1: çözümü engelleme eşek anti-fare 555 bağlı antikor 400 seyreltme 1 sağlayın. </li><li> Nemli bir oda içinde oda sıcaklığında 1 saat Aşama 4.9 ikincil antikor karışımı, 100 ul doku bölümleri inkübe edin. </li><li> Oda sıcaklığında her biri 5 dakika PBT ile üç kez dolu bir Coplin kavanoz doku bölümleri yıkayın. </li><li> Oda sıcaklığında 2 dakika boyunca tiramid sinyal amplifikasyonu tamponunda tiramid 200 seyreltme: 1 100 ul doku bölümleri yerleştirin. </li><li> Hemen PBT 5 dakika boyunca slaytlar her üç kez yıkanarak fazla tiramid çözüm kaldırmak. </li><li> dikkatliy aşırı PBT çıkarın ve hemen kapak bölümleri bir Montaj Orta bir damla (Malzeme Listesine bakınız). </li><li> Yavaşça doku kesitlerinde bir lamel yerleştirin ve hemen oje ile lamel mühür. </li><li> Mikroskopik incelemede önce birkaç saat 4 ° C&#39;de doku bölümleri saklayın. (- 40X büyütme 10) 405, 488 ve / veya 555 nm&#39;de floresan mikroskop altında inceleyin. </li></ol>

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The authors declare no conflict of interest.

Bazı dokularda 5MC oksitleme türevleri 5FC ve 5caC bildirilen düşük bolluk standart İmmünokimya protokolü için önemli sınırlamalar mevcut olsa da, peroksidaz-konjuge sekonder antikor dahil sabit doku ve hücrelerin bu sitozin modifikasyonların saptanmasına olanak (Şekil 2) . Ancak, tiramid çözümü ile optimum inkübasyon süresi sinyal yoğunluğu ve tiramid tabanlı sinyal amplifikasyon süresi arasında doğrusal bir ilişki ayrıntılar için, gözlenen tiramid sinyal amplifikasyon kiti her parti için deneysel optimize edilmelidir Almeida ve ark bakın. 2012 26 . Buna ek olarak, sinyal / arka plan oranı önemli ölçüde aşırı antikorların etkin şekilde çıkarılmasını sağlamak için bir Coplin kavanoza yıkama yaparak arttırılabilir. tiramid çözeltisi ile inkübasyondan sonra, hemen d PBT çözeltisi içinde yıkanarak reaksiyonu durdurmak önemlidirecrease plan boyaması. Bölümler işlem sırasında herhangi bir noktada kurumasına izin vermemek önemlidir. DNA, depürinasyon etkinliği 37 ° C sıcaklıkta depürinasyon reaksiyonun gerçekleştirilmesi ile geliştirilebilir. Bu çift sarmallı DNA ile özel olarak etkileşim edildiği gibi 4 N HCI kullanılarak yerine 2 birlikte, N, DAPI ile birlikte boyama izin değil, DNA depürinasyon 4 N HCI kullanılarak 5MC biçimlerinin değiştirilmiş biçimleriyle lekelenmesini artırır. Bu teknik, sitosin bazlar burada tespit modifiye formlarının güçlü bir yarı kantitatif bir değerlendirmesini sağlamak için birlikte, 5MC veya oksitleme türevleri mutlak düzeylerini değerlendirmek için kullanılamaz. Bu nedenle, diğer tamamlayıcı kullanılmasını öneririz ama niceliksel 16, 19 -24 yaklaşır. Memeli genomlarında 5MC oksitleme türevlerinin keşfinden beri çeşitli yaklaşımlar biyolojik rolleri 16, 19-24 çalışma geliştirilmiştir. Bu arayışlara rağmenches 5MC oksidasyon türevlerinin mutlak seviyelerini belirlemede değerli olabilir, onların uzaysal dağılımı 20, 26 ile ilgili bilgi vermemektedir. Biz başarıyla farklı hücre tiplerinde 5MC oksidasyon türevlerinin mekansal dağılımını ve yerelleştirme harita Burada anlatılan yöntemi kullandık gelişmekte olan ve yetişkin beynin 20 Mekansal dağılımı 20 ortaya çıkararak, bu teknik, biyolojik etkileri ve 5MC bu değiştirilmiş türevleri hücresel farklılaşma, geliştirme ve hastalık dahil tespit edilebilir çeşitli biyolojik bağlamlarda 5MC oksitlenmiş türevleri kaderini anlamak için önemli olabilir. Buna ek olarak, burada tarif yöntemi tyrami farklı inkübasyon sürelerindeki (boyama yoğunluğuna orantılı) peroksidaz reaksiyon kinetiği değerlendirerek farklı dokularda 5MC oksitlenmiş türevleri yarı-kantitatif değerlendirmeler verebilirDE 26.

DNA (5MC) sitozin bazlarının metilasyon transkripsiyonel 1 susturulması ile ilişkili omurgalıların genomları bulunan önemli bir epigenetik işareti temsil eder. 5MC tanıttı ve DNA methyltransferases 2-5 muhafaza ve genomik imprinting, X kromozomu inaktivasyonu, hücresel farklılaşma ve gelişme 3, 6. Sonuç olarak, bozulma dahil biyolojik süreçlerin bir dizi önemli rol oynamaktadır gösterilmiştir ediliyor 5MC genomik desenler hastalıklar 7, 8-11 bir dizi ile ilişkilidir. geliştirme ve hastalık 5MC rolünü anlamak ilerlemeye rağmen, hala bu işareti gelişmekte olan ve erişkin dokular çıkarıldı ediliyor ne ölçüde bilinmemektedir olduğunu. DNA, demetilasyon birçok potansiyel mekanizma en son aktif ve pasif demetilasyon mekanizmaları 12, 13, 14, 15 dahil olmak üzere, önerilmiştir. Discovery 1011 translokasyon enzim aracılı 5MC sıralı oksidasyon ürünleriES (tet1 / 2/3) gibi bunların ara maddeleri olarak hizmet edebilir olup 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5FC) ve DNA 16, 17, 18, ​​19 spekülasyonlar istendiğinde ökariyotik 5-carboxylcytosine (5caC) halinde kendi başlarına 13 DNA demetilasyon süreçleri ya da hareket olarak stabil epigenetik işaretler. Baz eksizyon onarım bileşeni, timin DNA glikozilaz (TDG) bağlamak ve DNA 19 den 5FC ve 5caC hem kaldırabilirsiniz gösteri, 20 aktif DNA demetilasyon modifiye 5MC türevleri bir rolü olduğunu iken. 5FC / 5caC transkripsiyonel düzenleme 29 bu markaların potansiyel tutulumu RNA II processivity noktalarının oranını modüle gösteren son kanıtlar. Nedeniyle 5MC oksitlenmiş formları bu potansiyel biyolojik önemi, biyokimyasal ve antikor bazlı tekniklerin bir dizi kendi genomik dağılımı ve küresel içerik 16, 19-24 çalışmak için istihdam edilmiştir. Verilen t en eminO 16-18, 20, 23, 25-27, farklı dokularda okside 5MC türevlerinin uzamsal dağılımını belirlemek deney önemli hale organlar, farklı hücre tiplerini içermektedir ve tadil edilmiş sitosin bazlar dağılımı dokusu ve hücre tipine özel düzenlenmiş olduğu omurgalı onların biyolojik fonksiyonları açıklanması için gerekli görev. Biyokimyasal ve antikor bazlı yaklaşımların çoğu farklı doku ve hücre tiplerinde 5MC biçimlerinin değiştirilmiş biçimleriyle dağıtımı ile ilgili bir mekansal bilgi sağlamaz. Buna karşılık, immunokimya tabanlı teknikler 5MC 28 ve oksitlenmiş türevleri 20 mekansal dağılımı ve nükleer lokalizasyonu değerlendirmek için hızlı bir araç sağlayabilir. Bu fare genomuna 18 (her 10 6 sitozin 3) bildirilen çok düşük 5FC bolluk (20 her 10 6 sitozin olarak) ve 5caC standart immünokimyanın için önemli bir sorun teşkil etmektedir, söylenir. İşte,Bu sağlam sağlayan son derece hassas immünokimyasal yöntem ve Memeli beyin dokusundaki sitozinin oksitlenmiş biçimde bir hızlı algılama tarif eder. Bir sinyal amplifikasyon aşaması ile birleştirilmiş peroksidaz-konjuge sekonder antikor ilave ederek, bu yöntem 5FC ve 5caC çok düşük miktarlarda tespit zorlukları aşılmaktadır. Buna ek olarak, bu teknik etkili bu epigenetik işaretlerin biyolojik fonksiyonları durulaştırmada diğer yaklaşımları tamamlayan, soy özel işaretleri ile sitozin birlikte tespit modifiye formları kullanılabilir.

<table><tbody><tr><td>Coplin jar</td><td>Cole-Parmer</td><td>UY-48585-30</td><td>Glass made</td></tr><tr><td>Xylene</td><td></td><td></td><td>Use only in Class II safety cabinet</td></tr><tr><td>Phosphate buffer saline (PBS)</td><td>Fisher Scientific</td><td>12821680</td><td>pH 7.5, filtered before use</td></tr><tr><td>4% paraformaldehyde (PFA)</td><td>Sigma Aldrich</td><td>P6148</td><td>Once made can be stored at -20 &amp;deg;C in aliquotes for several months</td></tr><tr><td>4% formaldehyde&amp;nbsp; (FA)</td><td>Sigma Aldrich</td><td>F8775</td><td>Once made can be stored at -20 &amp;deg;C in aliquotes for several months</td></tr><tr><td>Ethanol, anhydrous denatured, histological grade</td><td>Sigma Aldrich</td><td>64-17-5</td><td>95, 75, 50% in PBS</td></tr><tr><td>0.01% Tween 20 in PBS</td><td>Sigma Aldrich</td><td>P9416</td><td>PBT</td></tr><tr><td>0.5% Triton X-100 in PBS</td><td>Sigma Aldrich</td><td>X100</td><td>PBX</td></tr><tr><td>2 N HCl</td><td>Sigma Aldrich</td><td>71826</td><td>caution extremely toxic</td></tr><tr><td>100 mM Tris-HCl&amp;nbsp;</td><td>Promega</td><td>H5121</td><td>pH adjusted to 8.5</td></tr><tr><td>hydrophobic barrier pen</td><td>Abcam</td><td>ab2601</td><td>for immunohistochemistry</td></tr><tr><td>Slide moisture chamber</td><td>Scientific Device Laboratory</td><td>197-BL</td><td></td></tr><tr><td>anti-5mC mouse antibody</td><td>Diagenode</td><td>C15200081</td><td>monoclonal primary antibody</td></tr><tr><td>Anti-5hmC antibody</td><td>Active Motif</td><td>39791</td><td>rabbit polyclonal primary antibody</td></tr><tr><td>Anti-5caC antibody</td><td>Active Motif</td><td>61225</td><td>rabbit polyclonal primary antibody</td></tr><tr><td>Peroxidase-conjugated anti-rabbit seconday antibody</td><td>Dako</td><td>K1497</td><td></td></tr><tr><td>555-congjuated goat anti-mouse antibody</td><td>Molecular probes</td><td>A-11005</td><td>&amp;nbsp;secondary antibody</td></tr><tr><td>10% BSA</td><td>Sigma Aldrich</td><td>A9418</td><td>Blocking solution</td></tr><tr><td>22 x 32 mm Glass cover slips</td><td>BDH</td><td>406/0188/24</td><td></td></tr><tr><td>Tyramide Signal Amplification System</td><td>Perkin Elmer</td><td>NEL741001KT</td><td>Other fluorochome conjugated seconday antibodies can be used for co-detection of oxi-5mC&amp;nbsp;</td></tr><tr><td>&amp;nbsp;Mounting Medium with DAPI</td><td>Vector Labs</td><td>H-1200</td><td></td></tr><tr><td>Colourless nail polish</td><td></td><td></td><td></td></tr><tr><td>chicken polyclonal anti-GFAP polyclonal antibody</td><td>Thermo Scientific</td><td>PA5-18598</td><td>1:400 dilution</td></tr><tr><td>anti-NeuN mouse monoclonal antibody</td><td>Merck milipore</td><td>MAB377B</td><td>1:400 dilution</td></tr></tbody></table>

detection of modified forms of cytosine using sensitive immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Beyin dokusu bölümlerinde 5hmC dağılımını belirlemek için, özellikle bu işareti ile etkileşir, ancak ticari anti-5hmC antikoru kullanılarak, post-mitotik nöronlar, neun için bir marker ile epigenetik modifikasyon eş algılama gerçekleştirilmez değiştirilmiş formları ile sitosin 20, 25., yetişkin beyinde 5hmC ve 5caC dağılımları immünohistokimyasal analizi, önemli 5hmC boyama NeuN pozitif hücrelerle birlikte lokalize ise NeuN negatif glial hücreler genomik 5hmC (Şekil 1), 20 daha düşük seviyelerde sahip olduğunu ortaya koydu. Son zamanlarda Tet bağımlı 5MC oksidasyonu nöral kök hücre (NSC&#39;lerde) 20 soy tarifnamede esnasında etkin olduğunu göstermiştir. NSC&#39;lerde immünokimyasal belirlenemeyen olmasına rağmen, 5FC ve 5caC nöronal a doğru NSC&#39;lerde farklılaşma erken aşamalarında belirgin immün sergilemek<html> nd glial soy. Her iki işaretleri geçici erken nöronal ve glial farklılaşma 20 belirteçlerinin görünümü ile eş zamanlı olarak birikir. Bu glial farklılaşmanın başlaması için 3 gün NSC&#39;lerde sabit bir kültür glial markör GFAP ile işareti ko-lekeleme gerçekleştirildi NSCs ayırt 5caC dağılımını belirlemek. NSC&#39;lerde veya erişkin astrositlerin farklı olarak (veriler gösterilmemiştir), bu kültürlerde GFAP eksprese eden hücreler (Şekil 2), 20 nispeten büyük oranda güçlü 5caC sinyal görülmüştür. <img alt="Şekil 1" src="/files/ftp_upload/54416/54416fig1.jpg" /> 5hmC DAPI (mavi) ile zıt Yetişkin Beyin Doku Neun (kırmızı) ile (yeşil). Bireysel kanalları ve birleştirilmiş görünümü Şekil 1. Eş-immün gösterilmektedir. Ölçek çubukları 25 mikron bulunmaktadır.&quot;Blank&gt; Bu rakamın büyük halini görmek için lütfen buraya tıklayınız. <img alt="şekil 2" src="/files/ftp_upload/54416/54416fig2.jpg" /> Şekil Glial Farklılaşma Gün 3 de MGK Kültür GFAP ile 5caC 2. Eş-immün. Bireysel kanalları ve birleştirilmiş görünümü gösterilmektedir. Ölçek çubukları 20 mikron bulunmaktadır. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54416/54416fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry at JoVE.com

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Hassas immünohistokimyasal sitozin Modifiye Formlar Algılama

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

detection-modified-forms-cytosine-using-sensitive

Universidad San Sebastian

Research

JoVE Journal

Biology

7.2K Görüntüleme.  Université Libre de Bruxelles. Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Video: Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Immunostaining for DNA Modifications: Computational Analysis of Confocal Images

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of enzymatic oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) as well as detection of N6-methyladenine (6mA) in the DNA of multicellular organisms provided additional degrees of complexity to the epigenetic research. According to a growing body of experimental evidence, these novel DNA modifications may play specific roles in different cellular and developmental processes. Importantly, as some of these marks (e. g. 5hmC, 5fC and 5caC) exhibit tissue- and developmental stage-specific occurrence in vertebrates, immunochemistry represents an important tool allowing assessment of spatial distribution of DNA modifications in different biological contexts. Here the methods for computational analysis of DNA modifications visualized by immunostaining followed by confocal microscopy are described. Specifically, the generation of 2.5 dimension (2.5D) signal intensity plots, signal intensity profiles, quantification of staining intensity in multiple cells and determination of signal colocalization coefficients are shown. Collectively, these techniques may be operational in evaluating the levels and localization of these DNA modifications in the nucleus, contributing to elucidating their biological roles in metazoans.

Newly discovered oxidized forms of 5-methylcytosine (oxi-mCs), 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) may represent distinct DNA modifications with unique functional roles. Here a semi-quantitative workflow for visualization of oxi-mCs' spatial distribution, signal intensity profiling and colocalization is described.

DNA değişiklikler için Immunostaining: Confocal görüntülerin sayısal analiz

For several decades, 5-methylcytosine (5mC) has been thought to be the only DNA modification with a functional significance in metazoans. The discovery of ...

Genetik

High Sensitivity 5-hydroxymethylcytosine Detection in Balb/C Brain Tissue

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research. Mammalian DNA is enzymatically modified at the 5th carbon position of cytosine (C) residues to 5-mC, predominately in the context of CpG dinucleotides. 5-mC is amenable to enzymatic oxidation to 5-hmC by the Tet family of enzymes, which are believed to be involved in development and disease. Currently, the biological role of 5-hmC is not fully understood, but is generating a lot of interest due to its potential as a biomarker. This is due to several groundbreaking studies identifying 5-hydroxymethylcytosine in mouse embryonic stem (ES) and neuronal cells. Research techniques, including bisulfite sequencing methods, are unable to easily distinguish between 5-mC and 5-hmC . A few protocols exist that can measure global amounts of 5-hydroxymethylcytosine in the genome, including liquid chromatography coupled with mass spectrometry analysis or thin layer chromatography of single nucleosides digested from genomic DNA. Antibodies that target 5-hydroxymethylcytosine also exist, which can be used for dot blot analysis, immunofluorescence, or precipitation of hydroxymethylated DNA, but these antibodies do not have single base resolution.In addition, resolution depends on the size of the immunoprecipitated DNA and for microarray experiments, depends on probe design. Since it is unknown exactly where 5-hydroxymethylcytosine exists in the genome or its role in epigenetic regulation, new techniques are required that can identify locus specific hydroxymethylation. The EpiMark 5-hmC and 5-mC Analysis Kit provides a solution for distinguishing between these two modifications at specific loci. The EpiMark 5-hmC and 5-mC Analysis Kit is a simple and robust method for the identification and quantitation of 5-methylcytosine and 5-hydroxymethylcytosine within a specific DNA locus. This enzymatic approach utilizes the differential methylation sensitivity of the isoschizomers MspI and HpaII in a simple 3-step protocol. Genomic DNA of interest is treated with T4-BGT, adding a glucose moeity to 5-hydroxymethylcytosine. This reaction is sequence-independent, therefore all 5-hmC will be glucosylated; unmodified or 5-mC containing DNA will not be affected. This glucosylation is then followed by restriction endonuclease digestion. MspI and HpaII recognize the same sequence (CCGG) but are sensitive to different methylation states. HpaII cleaves only a completely unmodified site: any modification (5-mC, 5-hmC or 5-ghmC) at either cytosine blocks cleavage. MspI recognizes and cleaves 5-mC and 5-hmC, but not 5-ghmC. The third part of the protocol is interrogation of the locus by PCR. As little as 20 ng of input DNA can be used. Amplification of the experimental (glucosylated and digested) and control (mock glucosylated and digested) target DNA with primers flanking a CCGG site of interest (100-200 bp) is performed. If the CpG site contains 5-hydroxymethylcytosine, a band is detected after glucosylation and digestion, but not in the non-glucosylated control reaction. Real time PCR will give an approximation of how much hydroxymethylcytosine is in this particular site. In this experiment, we will analyze the 5-hydroxymethylcytosine amount in a mouse Babl/C brain sample by end point PCR.

The EpiMark 5-hmC and 5-mC Analysis Kit can be used to analyze and quantitate 5-methylcytosine and 5-hydroxymethylcytosine within a spe cific locus. The kit distinguishes 5-mC from 5-hmC by the addition of glucose to the hydroxyl group of 5-hmC via an enzymatic reaction utilizing &beta;-glucosyltransferase (T4-BGT). When 5-hmC occurs In the context of CCGG, this modification converts a cleavable MspI site to a non-cleavable site.

Balb / C Beyin Dokusu Yüksek Hassasiyet 5-hydroxymethylcytosine Algılama

DNA hydroxymethylation is a long known modification of DNA, but has recently become a focus in epigenetic research.   Mammalian DNA is enzymatically ...

Nörobilim

Immunohistochemical Detection of 5-Methylcytosine and 5-Hydroxymethylcytosine in Developing and Postmitotic Mouse Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a variety of human retinal degenerative diseases and pinpoint new treatment approaches. The nuclear architecture of mouse retina is organized in two different patterns: conventional and inverted. Conventional pattern is universal where heterochromatin is localized to the periphery of the nucleus, while active euchromatin resides in the nuclear interior. In contrast, inverted nuclear pattern is unique to the adult rod photoreceptor cell nuclei where heterochromatin localizes to the nuclear center, and euchromatin resides in the nuclear periphery. DNA methylation is predominantly observed in chromocenters. DNA methylation is a dynamic covalent modification on the cytosine residues (5-methylcytosine, 5mC) of CpG dinucleotides that are enriched in the promoter regions of many genes. Three DNA methyltransferases (DNMT1, DNMT3A and DNMT3B) participate in methylation of DNA during development. Detecting 5mC with immunohistochemical techniques is very challenging, contributing to variability in results, as all DNA bases including 5mC modified bases are hidden within the double-stranded DNA helix. However, detailed delineation of 5mC distribution during development is very informative. Here, we describe a reproducible technique for robust immunohistochemical detection of 5mC and another epigenetic DNA marker 5-hydroxymethylcytosine (5hmC), which colocalizes with the &#34;open&#34;, transcriptionally active chromatin in developing and postmitotic mouse retina.

The objective of this report is to describe the protocol for robust immunohistochemical detection of epigenetic markers, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in developing and postmitotic mouse retina.

5-metilsitozin ve gelişmekte olan 5-arasında bakterilerde görülen immunohistokimyasal tespiti ve Postmitotic fare Retina

The epigenetics of retinal development is a well-studied research field, which promises to bring a new level of understanding about the mechanisms of a ...

Gelişim Biyolojisi

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Floresan Nöronal Dokularda Kalıcı DNA Virüs Genomu ve Transkriptlerinin Algılama In situ Melezleme immün ile birlikte

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

Her Yerde Bulunan, SUMOile Edilmiş ve ADP-Ribosile Edilmiş DNA-Protein Çapraz Bağlarının Ölçülmesi

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

Yazar Spotlight: DNA Protein Çapraz Bağlarının Kantitatif Tespiti ve Translasyon Sonrası Modifikasyonları  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-Protein Çapraz Bağlarının Kantitatif Tespiti ve Translasyon Sonrası Modifikasyonları

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Biyokimya

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

Genomik DNA 5-hydroxymethylcytosine seçici Yakalama

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

Biyoloji

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Farelerin Nöral Kök Hücreleri ve Beyinlerinde 5-Hidroksimetilsitozozin Insaptama

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&#38;E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&#38;E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

QPCR Formalinle sabit, Parafin-gömülü Biyopsi Doku sitomegaloviral DNA tespiti için bir Hassas ve Hızlı Yöntem mi

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for ...

Hassas immünohistokimyasal sitozin Modifiye Formlar Algılama

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Genomik DNA 5-hydroxymethylcytosine seçici Yakalama

Farelerin Nöral Kök Hücreleri ve Beyinlerinde 5-Hidroksimetilsitozozin Insaptama

QPCR Formalinle sabit, Parafin-gömülü Biyopsi Doku sitomegaloviral DNA tespiti için bir Hassas ve Hızlı Yöntem mi