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>

<ol>
	<li>Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methylation+patterns+and+epigenetic+memory.">DNA methylation patterns and epigenetic memory.</a> Genes Dev. 16, 6-21 (2002).</li><li>Reik, W., Dean, W., Walter, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+reprogramming+in+mammalian+development.">Epigenetic reprogramming in mammalian development.</a> Science. 293, 1089-1093 (2001).</li><li>Jaenisch, R., Bird, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+regulation+of+gene+expression:+how+the+genome+integrates+intrinsic+and+environmental+signals.">Epigenetic regulation of gene expression: how the genome integrates intrinsic and environmental signals.</a> Nat Genet. 33, 245-254 (2003).</li><li>Li, E., Bestor, T. H., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutation+of+the+DNA+methyltransferase+gene+results+in+embryonic+lethality.">Targeted mutation of the DNA methyltransferase gene results in embryonic lethality.</a> Cell. 69 (6), 915-926 (1992).</li><li>Okano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+methyltransferases+Dnmt3a+and+Dnmt3b+are+essential+for+de+novo+methylation+and+mammalian+development.">DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development.</a> Cell. 99 (3), 247-257 (1999).</li><li>Cedar, H., Bergman, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Programming+of+DNA+methylation+patterns.">Programming of DNA methylation patterns.</a> Annu Rev Biochem. 81, 97-117 (2012).</li><li>Amir, R. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rett+syndrome+is+caused+by+mutations+in+X-linked+MECP2,+encoding+methyl-CpG-binding+protein+2.">Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2.</a> Nat Genet. 23, 185-188 (1999).</li><li>Chouliaras, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consistent+decrease+in+global+DNA+methylation+and+hydroxymethylation+in+the+hippocampus+of+Alzheimer's+disease+patients.">Consistent decrease in global DNA methylation and hydroxymethylation in the hippocampus of Alzheimer's disease patients.</a> Neurobiol Aging. 34, 2091-2099 (2013).</li><li>Jowaed, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methylation+regulates+alpha-synuclein+expression+and+is+decreased+in+Parkinson's+disease+patients'+brains.">Methylation regulates alpha-synuclein expression and is decreased in Parkinson's disease patients' brains.</a> J Neurosci. 30, 6355-6359 (2010).</li><li>Reik, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+at+onset+in+Huntington's+disease+and+methylation+at+D4S95.">Age at onset in Huntington's disease and methylation at D4S95.</a> J Med Genet. 30, 185-188 (1993).</li><li>Landgrave-G&#242;mez, J., Mercado-G&#242;mez, O., Guevara-Guzm&#225;n, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+mechanisms+in+neurological+and+neurodegenerative+diseases.">Epigenetic mechanisms in neurological and neurodegenerative diseases.</a> Front Cell Neurosci. 27, 9-58 (2015).</li><li>Seisenberger, S., Peat, J. R., Hore, T. A., Santos, F., Dean, W., Reik, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+DNA+methylation+in+the+mammalian+life+cycle:+building+and+breaking+epigenetic+barriers.">Reprogramming DNA methylation in the mammalian life cycle: building and breaking epigenetic barriers.</a> Philos Trans R Soc Lond B Biol Sci. 368 (1609), (2013).</li><li>Wu, H., Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+and+functions+of+Tet+protein-mediated+5-+methylcytosine+oxidation.">Mechanisms and functions of Tet protein-mediated 5- methylcytosine oxidation.</a> Genes Dev. 25, 2436-2452 (2011).</li><li>Oswald, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+demethylation+of+the+paternal+genome+in+the+mouse+zygote.">Active demethylation of the paternal genome in the mouse zygote.</a> Curr Biol. 10 (8), 475-478 (2000).</li><li>Ooi, S. K., Bestor, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+colorful+history+of+active+DNA+demethylation.">The colorful history of active DNA demethylation.</a> Cell. 133 (7), 1145-1148 (2008).</li><li>Kriaucionis, S., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nuclear+DNA+base+5-hydroxymethylcytosine+is+present+in+Purkinje+neurons+and+the+brain.">The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain.</a> Science. 324, 929-930 (2009).</li><li>Tahiliani, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conversion+of+5-methylcytosine+to+5-hydroxymethylcytosine+in+mammalian+DNA+by+MLL+partner+TET1.">Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1.</a> Science. 324, 930-935 (2009).</li><li>Ito, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet+proteins+can+convert+5-methylcytosine+to+5-formylcytosine+and+5-carboxylcytosine.">Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine.</a> Science. 333, 1300-1303 (2011).</li><li>He, Y. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet-mediated+formation+of+5-carboxylcytosine+and+its+excision+by+TDG+in+mammalian+DNA.">Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA.</a> Science. 333, 1303-1307 (2011).</li><li>Wheldon, L. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+accumulation+of+5-carboxylcytosine+indicates+involvement+of+active+demethylation+in+lineage+specification+of+neural+stem+cells.">Transient accumulation of 5-carboxylcytosine indicates involvement of active demethylation in lineage specification of neural stem cells.</a> Cell Rep. 7, 1353-1361 (2014).</li><li>Yu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Base-resolution+analysis+of+5-hydroxymethylcytosine+in+the+mammalian+genome.">Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.</a> Cell. 149, 1368-1380 (2012).</li><li>Lu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+modification+assisted+bisulfite+sequencing+(CAB-Seq)+for+5-carboxylcytosine+detection+in+DNA.">Chemical modification assisted bisulfite sequencing (CAB-Seq) for 5-carboxylcytosine detection in DNA.</a> J Am Chem Soc. 26, 9315-9317 (2013).</li><li>Globisch, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+distribution+of+5-hydroxymethylcytosine+and+search+for+active+demethylation+intermediates.">Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates.</a> PLoS One. 5, e15367 (2010).</li><li>Szwagierczak, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+enzymatic+quantifi+cation+of+5-+hydroxymethylcytosine+in+genomic+DNA.">Sensitive enzymatic quantifi cation of 5- hydroxymethylcytosine in genomic DNA.</a> Nucleic Acids Res. 38, e181 (2010).</li><li>Ruzov, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage-specific+distribution+of+high+levels+of+genomic+5-hydroxymethylcytosine+in+mammalian+development.">Lineage-specific distribution of high levels of genomic 5-hydroxymethylcytosine in mammalian development.</a> Cell Res. 21, 1332-1334 (2011).</li><li>Almeida, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semi-quantitative+immunohistochemical+detection+of+5-hydroxymethyl-cytosine+reveals+conservation+of+its+tissue+distribution+between+amphibians+and+mammals.">Semi-quantitative immunohistochemical detection of 5-hydroxymethyl-cytosine reveals conservation of its tissue distribution between amphibians and mammals.</a> Epigenetics. 7, 137-140 (2012).</li><li>Lister, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epigenomic+reconfiguration+during+mammalian+brain+development.">Global epigenomic reconfiguration during mammalian brain development.</a> Science. 341, 1237905 (2013).</li><li>Santos, F., Dean, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+11,+Using+immunofluorescence+to+observe+methylation+changes+in+mammalian+preimplantation+embryos.">Chapter 11, Using immunofluorescence to observe methylation changes in mammalian preimplantation embryos.</a> Nuclear reprogramming methods and Protocols. , 129-138 (2006).</li><li>Wang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+for+5-carboxycytosine+recognition+by+RNA+polymerase+II+elongation+complex.">Molecular basis for 5-carboxycytosine recognition by RNA polymerase II elongation complex.</a> Nature. 523, 621-625 (2015).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="1281">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:388px;">Coplin jar</td>
			<td style="width:180px;">Cole-Parmer</td>
			<td style="width:119px;">UY-48585-30</td>
			<td style="width:595px;">Glass made</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylene</td>
			<td></td>
			<td></td>
			<td>Use only in Class II safety cabinet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate buffer saline (PBS)</td>
			<td>Fisher Scientific</td>
			<td>12821680</td>
			<td>pPH 7.5, filtered before use</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% paraformaldehyde (PFA)</td>
			<td>Sigma Aldrich</td>
			<td>P6148</td>
			<td>Once made can be stored at -20&#176;C in aliquotes for several months</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">4% formaldehyde&#160; (FA)</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td>Once made can be stored at -20&#176;C in aliquotes for several months</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, anhydrous denatured, histological grade</td>
			<td>Sigma Aldrich</td>
			<td>&#160;64-17-5&#160;&#160;</td>
			<td>95, 75, 50 % in PBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.01 % Tween 20 in PBS</td>
			<td>Sigma Aldrich</td>
			<td>P9416</td>
			<td>PBT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.5% Triton X-100 in PBS</td>
			<td>Sigma Aldrich</td>
			<td>X100</td>
			<td>PBX</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2 N HCl</td>
			<td>Sigma Aldrich</td>
			<td>71826</td>
			<td>caution extremely toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 mM Tris-HCl&#160;</td>
			<td>Promega</td>
			<td>H5121</td>
			<td>PH adjusted to 8.5</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">hydrophobic barrier pen</td>
			<td>Abcam</td>
			<td>ab2601</td>
			<td>for immunohistochemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Slide moisture chamber</td>
			<td>Scientific Device Laboratory</td>
			<td>197-BL</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">anti-5mC mouse antibody</td>
			<td>Diagenode</td>
			<td>C15200081</td>
			<td>monoclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-5hmC antibody</td>
			<td>Active Motif</td>
			<td>39791</td>
			<td>rabbit polyclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Anti-5caC antibody</td>
			<td>Active Motif</td>
			<td>61225</td>
			<td>rabbit polyclonal primary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Peroxidase-conjugated anti-rabbit seconday antibody</td>
			<td>Dako</td>
			<td>K1497</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">555-congjuated goat anti-mouse antibody</td>
			<td>Molecular probes</td>
			<td>A-11005</td>
			<td>&#160;secondary antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10% BSA</td>
			<td>Sigma Aldrich</td>
			<td>A9418</td>
			<td>Blocking solution</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">22x32mm Glass cover slips</td>
			<td>BDH</td>
			<td>406/0188/24</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">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&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;Mounting Medium with DAPI</td>
			<td>Vector Labs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Colourless nail polish</td>
			<td style="width:180px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chicken polyclonal anti-GFAP polyclonal antibody</td>
			<td>Thermo Scientific</td>
			<td style="width:119px;">PA5-18598</td>
			<td>1:400 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-NeuN mouse monoclonal antibody</td>
			<td>Merck milipore</td>
			<td style="width:119px;">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/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

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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

Thin Sectioning of Slice Preparations for Immunohistochemistry

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been preserved, freshly removed, or excised but kept viable, as in slice preparations of brain tissue.  Subsequent microscopic studies of this material can be challenging, as the tissue samples may be difficult to handle.  Demonstrated here is a method for obtaining thin cryostat sections of tissue with a thickness that may range from 0.2-5.0 mm.  We routinely cut 400 micron thick Vibratome brain slices serially into 5-10 micron coronal cryostat sections.  The slices are typically first used for electrophysiology experiments and then require microscopic analysis of the cytoarchitecture of the region from which the recordings were observed.  We have constructed a simple device that allows controlled and reproducible preparation and positioning of the tissue slice.  This device consists of a cylinder 5 cm in length with a diameter of 1.2 cm, which serves as a freezing stage for the slice.  A ring snugly slides over the cylinder providing walls around the slice allowing the tissue to be immersed in freezing compound (e.g., OCT).  This is then quickly frozen with crushed dry ice and the resulting  wafer  can be position easily for cryostat sectioning.  Thin sections can be thaw-mounted onto coated slides to allow further studies to be performed, such as various staining methods, in situ hybridization, or immunohistochemistry, as demonstrated here.

The present method allows reproducible cryostat sectioning of small, difficult-to-manage, tissue pieces, such as biopsies and brain slices. We utilize a simple aluminum freezing stage to facilitate handling of tissue and a standard cryostat to routinely produce 5-10 micron serial sections from 400 micron thick brain slices.

Dilim İmmünohistokimya için hazırlıklar İnce Kesit

Many investigations in neuroscience, as well as other disciplines, involve studying small, yet macroscopic pieces or sections of tissue that have been ...

Biyoloji

Preventing the Spread of Malaria and Dengue Fever Using Genetically Modified Mosquitoes

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced, as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission. Population replacement strategy, the idea that transgenic mosquitoes can be released into the wild to control disease transmission, is introduced as well as the concept of genetic drive and the design criterion for an effective genetic drive system. The ethical considerations of releasing genetically-modified organisms into the wild are also discussed.

Genetiği Değiştirilmiş Sivrisinekler kullanarak Sıtma ve Dang Ateşi Yayılması Önleme

In this candid interview, Anthony A. James explains how mosquito genetics can be exploited to control malaria and dengue transmission.   Population ...

Immunohistochemistry: Paraffin Sections Using the Vectastain ABC Kit from Vector Labs

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific antibodies. There are two methods: the direct and indirect method. In this experiment, we will only describe the use of indirect IHC staining. Indirect IHC staining utilizes highly specific primary and biotin-conjugated secondary antibodies. Primary antibodies are utilized to discretely identify proteins of interest by binding to a specific epitope, while secondary antibodies subtract for non-specific background staining and amplify signal by forming complexes to the primary antibody. Slides can either be generated from frozen sections, or paraffin embedded sections mounted on glass slides. In this protocol, we discuss the preparation of paraffin-embedded sections by dewaxing, hydration using an alcohol gradient, heat induced antigen retrieval, and blocking of endogenous peroxidase activity and non-specific binding sites. Some sections are then stained with antibodies specific for T cell marker CD8 and while others are stained for tyrosine hydroxylase. The slides are subsequently treated with appropriate secondary antibodies conjugated to biotin, then developed utilizing avidin-conjugated horseradish peroxidase (HRP) with Diaminiobenzidine (DAB) as substrate. Following development, the slides are counterstained for contrast, and mounted under coverslips with permount. After adequate drying, these slides are then ready for imaging.

İmmünohistokimya: Parafin Bölüm Vektör Labs Vectastain ABC Seti

Immunohistochemistry (IHC) is a valuable technique utilized to localize/visualize protein expression in a mounted tissue section using specific ...

Immunohistochemistry on Paraffin Sections of Mouse Epidermis Using Fluorescent Antibodies

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, suprabasal, and stratum corneum layers. The precise localization within the epidermis of a particular protein lends clues toward its functional role within the epidermis. In this chapter, we describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory. Paraffin sections generally provide much better morphology, hence, superior results and photographs; however, not all antibodies will work with the harsh fixation and treatment involved in their processing. Therefore, the protocol for frozen sectioning is also included. Within paraffin sectioning, two fixation protocols are described (Bouin's and paraformaldehyde); the choice of fixative will be directly related to the antibody specifications and may require another fixing method.

We describe a reliable method for immunolocalization within the epidermis modified for both frozen and paraffin sections that we use very routinely in our laboratory.

Floresan Antikorlar Fare Epidermis Parafin Bölüm İmmünohistokimya

In the epidermis, immunohistochemistry is an efficient means of localizing specific proteins to their relative expression compartment; namely the basal, ...

Drosophila Larval NMJ Immunohistochemistry

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 31 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. Immunohistochemistry can be used to clearly image the components of the NMJ. In this article, we demonstrate how to use antibody staining to visualize the Drosophila larval NMJ.

Drosophila Larval NMJ Immunohistochemistry

This protocol demonstrates how to perform immunohistochemistry on dissected Drosophila larva.

Drosophila Larvaların NMJ İmmünohistokimya

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Optical Recording of Electrical Activity in Guinea-pig Enteric Networks using Voltage-sensitive Dyes

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons (10 to 25 &mu;m in diameter) are arranged in plexuses that are confined to distinct planes of the gut wall 1; the myenteric plexus can be found between the longitudinal and circular muscle layers, and the submucous plexus between the circular muscle layer and the mucosa. Since the effector systems for these plexuses (transporting epithelium, endocrine cells, immune elements, blood vessels and smooth muscle) are also contained within the gut wall, semi-intact preparations can be dissected that preserve individual components of different reflex pathways. The behavior of the effector systems is controlled by the submucous and myenteric plexuses acting in concert. Therefore, detailed knowledge of synaptic interactions within and between ganglia, and of communication between the plexuses, is essential for understanding normal gastrointestinal function. The ENS, as an intact nervous system, is a unique experimental model in which one can correlate molecular and cellular events with the electrical behavior of the neuronal network and its physiological outputs. Because of the quasi-two-dimensional organization of its plexuses, the ENS is particularly well suited for the study of neural networks using multiple site optical recording techniques that employ voltage-sensitive dyes 2,7,8,9. We will illustrate here the use of a relatively new naphthylstyryl-pyridinium dye (di-4-ANEPPDHQ) 3 that offers multiple advantages over its predecessors, including very low phototoxicity, slow rate of internalization, and remarkable chemical stability. When used in conjunction with a camera that permits sub-millisecond time resolution, this dye allows us to monitor the electrical activity of all the neurons in the field of view with a maximal spatial resolution of ~ 2.5 &mu;m at 100X magnification. At lower magnification (10X or 20X), the sacrifice of single-cell resolution is compensated by a gain in perspective, revealing the intricacies of the inter-ganglionic circuitry.

This protocol illustrates how voltage-sensitive dyes enable optical recording of electrical activity from intact neural networks such as the plexuses of the guinea-pig enteric nervous system, with an adjustable resolution that ranges from single-cells to multi-ganglionic circuitry.

Gerilim duyarlı Boyalar kullanarak Gine domuzu Enterik Ağları Elektrik Faaliyet Optik Kayıt

The enteric nervous system (ENS) is a self-contained network with identified functions, capable of performing complex behaviors in isolation. Its neurons ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

Drosophila pupa Karın İmmünohistokimya

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

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 ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

Turnusol Testi kullanarak Bakterilerin Kolorimetrik Tespiti

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Sensitive Measurement of Mitophagy by Flow Cytometry Using the pH-dependent Fluorescent Reporter mt-Keima

Mitophagy is a process of selective removal of damaged or unnecessary mitochondria using autophagy machinery. Close links have been found between defective mitophagy and various human diseases, including neurodegenerative diseases, cancer, and metabolic diseases. In addition, recent studies have shown that mitophagy is involved in normal cellular processes, such as differentiation and development. However, the precise role of and molecular mechanisms underlying mitophagy require further study. Therefore, it is critical to develop a robust and convenient method for measuring changes in mitophagy activity. Here, we describe a detailed protocol for quantitatively assessing mitophagy activity through flow cytometry using the mitochondria-targeted fluorescent protein Keima (mt-Keima). This flow cytometry assay can analyze mitophagy activity more rapidly and sensitively than conventional microscopy- or immunoblotting-based methods. This protocol can be applied to analyze mitophagy activity in various cell types.

Mitophagy, the selective degradation of mitochondria, has been implicated in mitochondrial homeostasis and is deregulated in various human diseases. However, convenient experimental methods for measuring mitophagy activity are very limited. Here, we provide a sensitive assay for measuring mitophagy activity using flow cytometry.