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.).

Tutte le procedure di origine animale coinvolti sono stati eseguiti in accordo con l&#39;Università di Nottingham di bordo esame etico. 1. Selezione Preparazione del tessuto adatto per Immunocolorazione <ol><li> Genera sezione di paraffina-embedded di wild-type embrioni CD1 del mouse e tessuti adulti cerebrali come descritto in precedenza 25. Utilizzare sezioni di tessuto cerebrale fisse sia con il 4% di formaldeide (FA) o 4% paraformaldeide (PFA) per il metodo di 25 immunocolorazione. NOTA: L&#39;uso di sezioni di tessuto paraffina richiede de-ceretta prima di etichettatura degli anticorpi. Dal momento che il trattamento con 2-4 M di acido cloridrico (HCl) impiegato nel protocollo per la denaturazione del DNA non è compatibile con la maggior parte delle strategie di recupero dell&#39;antigene, si consiglia l&#39;uso di una sezione cryo- o microtomo per co-rilevazione dei derivati ​​di ossidazione 5MC con proteine marcatori. </li></ol> 2. deparaffinazione paraffina del tessuto integrato Sezioni &lt;<html> / P&gt; <ol><li> In una cabina di sicurezza classe in esecuzione II, lavare la paraffina sezioni di tessuto incorporati in una vaschetta Coplin piena di xilene, 2 volte per 10 minuti ciascuno a temperatura ambiente. NOTA: E &#39;importante utilizzare xilene fresco come la rimozione della paraffina incompleta può portare a pattern di colorazione incoerenti. </li><li> Dopo deceratura, rapidamente reidratare le sezioni di tessuto di lavaggio consecutivamente in 95, 75, e 50% di etanolo per 10 min ciascuno a RT. </li></ol> 3. Fissazione e permeabilizzazione di Cryo- e Sezioni microtomo <ol><li> Fissare le sezioni di tessuto reidratati mettendoli in entrambi ghiacciata 4% PFA o 4% FA per 15 minuti a RT. </li><li> Rimuovere l&#39;eccesso fissativo lavando sezioni in PBS (Phosphate Buffer saline) per 5 minuti a temperatura ambiente. </li><li> Permeabilize le sezioni di tessuto posizionando in una vaschetta Coplin riempita con PBX (0,5% Triton X-100 in PBS) per 30 minuti a RT. Rimuovere l&#39;eccesso PBX lavando sezioni a breve in PBT (0,01% di Tween 20 in PBS). </li></ol>jove_title &quot;&gt; 4. Immunocolorazione per ossi-5MC Derivati <ol><li> Posizionare le sezioni permeabilizzate in 2 N HCl per 60 minuti a temperatura ambiente per depurinazione del DNA. NOTA: Anche se alte concentrazioni di HCl (ad esempio, 4 N) portare ad una più efficiente denaturazione del DNA, che non sono compatibili per la co-rilevamento di ossi-mC con DAPI. </li><li> Posizionare le sezioni in 10 mM Tris-HCl (pH 8,5) per 30 minuti a RT per neutralizzare il HCl. Alternativamente, lavare sezioni tre volte per 5 minuti ciascuno in PBS. Incubare le sezioni in PBT per 5 minuti a temperatura ambiente. </li><li> Rimuovere con attenzione il liquido dalla zona circostante la sezione di tessuto senza lasciare che la sezione di tessuto per asciugare completamente in qualsiasi fase agitando delicatamente il vetrino. Utilizzare una penna barriera idrofobica a circondare la sezione senza toccare la sezione. NOTA: La penna barriera idrofobica riduce il volume di anticorpo necessaria per macchiare il tessuto e può consentire più sezioni da colorate con different anticorpi sullo stesso vetrino. </li><li> Incubare sezioni in 100 ml di soluzione bloccante (10% di albumina di siero bovino in PBS) per 1 ora a RT in una camera umida. NOTA: Saltare questo passaggio non inciderebbe l&#39;efficienza di macchiare le basi citosina modificati. </li><li> Incubare le sezioni di tessuto in 100 ml di una diluizione 1: 5.000 del topo monoclonale anti-5hmC e una diluizione 1: 1.000 del policlonale di coniglio anti-5caC anticorpi primari nel bloccare soluzione per 1 ora a temperatura ambiente in una camera umida. In alternativa, eseguire la notte di incubazione a 4 ° C, se necessario. NOTA: Le sezioni processati senza anticorpi primari possono servire come idonei controlli negativi per la procedura di colorazione. I 12,5 giorni dopo coitum murine sezioni del cervello embrionali arricchito in 5caC, 5FC e 5hmC 20 può essere utilizzato come controllo positivo. </li><li> Rimuovere gli anticorpi in eccesso lavando sezioni in una vaschetta Coplin piena di PBT tre volte per 5 minuti ciascuno in una Copbarattolo lin a temperatura ambiente. NOTA: L&#39;aumento del volume di soluzioni di lavaggio può ridurre la colorazione di fondo. </li><li> Rimuovere l&#39;eccesso PBT e, se necessario, circondare sezioni di nuovo con la penna barriera idrofobica come PBT contiene detergenti che possono indebolire la barriera idrofobica. </li><li> Effettuare una diluizione 1: 400 di capra anti-coniglio anticorpo HRP-coniugato e una diluizione 1: 400 di asino anti-topo anticorpi 555 coniugato nel bloccare soluzione. </li><li> Incubare le sezioni di tessuto in 100 ml di miscela anticorpo secondario dal punto 4.9 per 1 ora a temperatura ambiente in una camera umida. </li><li> Lavare le sezioni di tessuto in una vaschetta Coplin piena di PBT tre volte per 5 minuti ciascuno a RT. </li><li> Posizionare le sezioni di tessuto in 100 ml di una diluizione 1: 200 di tiramide nel buffer amplificazione del segnale tiramide per 2 minuti a temperatura ambiente. </li><li> Immediatamente, rimuovere la soluzione in eccesso tiramide lavando i vetrini tre volte per 5 minuti ciascuno in PBT. </li><li> carefully rimuovere l&#39;eccesso di PBT e coprire immediatamente le sezioni con una goccia di un supporto di montaggio (vedi elenco dei materiali). </li><li> Delicatamente mettere un vetrino sulle sezioni di tessuto e sigillare immediatamente il coprioggetto con smalto. </li><li> Conservare le sezioni di tessuto a 4 ° C per diverse ore prima dell&#39;esame microscopico. Esaminare al microscopio a fluorescenza a 405, 488 e / o 555 nm (10 - 40X di ingrandimento). </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.

Sebbene la bassa abbondanza riferito derivati ​​ossidazione 5MC, 5FC e 5caC in alcuni tessuti potrebbe presentare limitazioni significative per un protocollo immunochimica standard, l&#39;incorporazione di anticorpi secondari coniugati con perossidasi ha permesso la rilevazione di tali modifiche citosina in tessuti e cellule fissate (Figura 2) . Tuttavia, il tempo ottimale di incubazione con la soluzione tiramide deve essere ottimizzata sperimentalmente per ciascun lotto di tiramide kit di amplificazione del segnale in cui viene osservata una relazione lineare tra l&#39;intensità del segnale e la durata di amplificazione del segnale basato tiramide, per i dettagli si riferiscono a Almeida et al. 2012 26 . Inoltre, il rapporto segnale / sfondo può essere notevolmente migliorata portando i lavaggi in una vaschetta Coplin per consentire la rimozione efficiente di anticorpi in eccesso. Dopo l&#39;incubazione con la soluzione tiramide, è importante interrompere immediatamente la reazione mediante lavaggio in soluzione PBT a dsfondo ecrease colorazione. È fondamentale non permettere alle sezioni asciugare in qualsiasi momento durante la procedura. L&#39;efficienza di depurinazione DNA può essere migliorata effettuando la reazione depurinazione a 37 ° C. Durante l&#39;utilizzo di 4 N HCl invece di 2 N migliora la colorazione delle forme modificate di 5MC utilizzando 4 N HCl per depurinazione DNA non avrebbe permesso di co-colorazione con DAPI, come interagisce esclusivamente con DNA a doppio filamento. Sebbene questa tecnica può fornire solide analisi semi-quantitativa delle forme modificate di basi citosina dove rilevabile, non può essere utilizzato per valutare i livelli assoluti di 5MC o suoi derivati ​​di ossidazione. Pertanto, si consiglia l&#39;uso di altri complementari ma quantitativa approcci 16, 19 -24. Dalla scoperta del 5MC derivati ​​di ossidazione nei genomi dei mammiferi, diversi approcci sono stati sviluppati per studiare i loro ruoli biologici 16, 19-24. Anche se questi avvicinamenches possono essere utili nel determinare i livelli assoluti di derivati ​​di ossidazione 5MC, non forniscono informazioni sulle loro distribuzione spaziale 20, 26. Abbiamo usato con successo il metodo descritto qui per mappare la distribuzione spaziale e la localizzazione dei derivati ​​di ossidazione 5MC in diversi tipi di cellule di sviluppo e cervello adulto 20 Rivelando la loro distribuzione spaziale 20, questa tecnica può essere fondamentale per comprendere le implicazioni biologiche e il destino dei derivati ​​ossidati di 5MC in vari contesti biologici in cui questi derivati ​​modificati di 5MC possono essere rilevati tra cui cellulari differenziazione, lo sviluppo e la malattia. Inoltre, il metodo che descriviamo qui può dare valutazioni semi-quantitativa dei derivati ​​ossidati di 5MC in diversi tessuti valutando la cinetica di reazione perossidasi (che è proporzionale alla intensità di colorazione) a differenti tempi di incubazione con tyramide 26.

La metilazione delle basi citosina nel DNA (5MC) rappresenta un segno importante epigenetica trovato nei vertebrati genoma associate a trascrizionale silenziamento 1. 5MC viene introdotto e mantenuto da DNA metiltransferasi 2-5, e ha dimostrato di giocare un ruolo importante in molti processi biologici tra cui imprinting genomico, inattivazione del cromosoma X, differenziazione cellulare, e sviluppo 3, 6. Di conseguenza, l&#39;interruzione della 5MC modelli genomici è associato con un certo numero di malattie 7, 8-11. Nonostante i progressi nella comprensione del ruolo 5MC nello sviluppo e nella malattia, è ancora rimangono in gran parte sconosciuto come questo marchio è stato rimosso in via di sviluppo e tessuti adulti. Diversi meccanismi potenziali di demetilazione DNA sono stati recentemente proposti compresi meccanismi attivi e passivi demetilazione 12, 13, 14, 15. Scoperta dei prodotti di ossidazione 5MC sequenziale mediate da TEN-eleven traslocazione enzymES (tet1 / 2/3) come il 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5FC), e 5-carboxylcytosine (5caC) in eucarioti DNA 16, 17, 18, ​​19 ha spinto le speculazioni se essi possono servire come intermedi di processi demetilazione del DNA o segni epigenetici agire come stabili nel loro diritto 13. Mentre la dimostrazione che la componente di base di riparazione per escissione, glicosilasi timina-DNA (TDG) può legare e rimuovere sia 5FC e 5caC dal DNA 19, 20 suggerisce un ruolo per i derivati ​​5MC modificati in una demetilazione del DNA attiva. Recenti evidenze che dimostrano che 5FC / 5caC in grado di modulare il tasso di punti processività RNA II al potenziale coinvolgimento di questi marchi nella regolazione trascrizionale 29. A causa di questo potenziale importanza biologica delle forme ossidate di 5MC, una serie di tecniche biochimiche e anticorpi base sono stati impiegati per studiare la loro distribuzione genomica e contenuti globali 16, 19-24. Dato che la maggior parte di tha vertebrato organi sono costituiti da diversi tipi di cellule e che la distribuzione delle basi citosina modificati è tessuto e cellula-specifico tipo 16-18, 20, 23, 25-27, determinare la distribuzione spaziale dei derivati ​​5MC ossidati in diversi tessuti diventa un importante sperimentale compito necessario per svelare le loro funzioni biologiche. La maggior parte degli approcci biochimici e anticorpi base non forniscono alcuna informazione territoriale sulla distribuzione delle forme modificate di 5MC nel tessuto diverso e tipi di cellule. Al contrario, le tecniche di immunochimica possono fornire uno strumento rapida per valutare la distribuzione spaziale e localizzazione nucleare di 5MC 28 e suoi derivati ​​ossidato 20. Che si dice, l&#39;riportate molto bassa abbondanza di 5FC (20 in ogni 10 6 citosina) e 5caC (3 in ogni 10 6 citosina) nel genoma del topo 18 rappresenta una sfida significativa per immunochimica standard. Qui,si descrive un metodo immunochimico altamente sensibile che fornisce robustezza ed una rapida individuazione della forma ossidata di citosina nel tessuto cerebrale dei mammiferi. Incorporando anticorpi secondari coniugati con perossidasi accoppiato con un passo di amplificazione del segnale, questo metodo aggira le sfide di rilevare le quantità molto basse di 5FC e 5caC. Inoltre, questa tecnica può essere utilizzata per co-rilevare le forme modificate di citosina con marcatori specifici lignaggio, integrando efficacemente altri approcci a chiarire le funzioni biologiche di questi segni epigenetici.

<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.

Per determinare la distribuzione delle 5hmC in sezioni di tessuto cerebrale, abbiamo eseguito co-rivelazione di questa modifica epigenetica con un marcatore per i neuroni post-mitotici, NeuN, impiegando l&#39;anticorpo commerciale anti-5hmC che interagisce specificamente con questo marchio, ma non con altre forme di modifica citosina 20, 25. l&#39;analisi immunoistochimica di 5hmC e 5caC distribuzioni nel cervello adulto ha rivelato che, mentre il primo piano colorazione 5hmC co-localizza con cellule positive NeuN, cellule gliali NeuN-negativi possiedono bassi livelli di genomica 5hmC (Figura 1) 20. Abbiamo recentemente dimostrato che Tet-dipendente ossidazione 5MC è operativo durante la specifica stirpe di cellule staminali neurali (NSC) 20. Pur essendo immunochimicamente rilevabile nel NSC, 5FC e 5caC esibiscono immunostaining di primo piano nelle fasi iniziali della differenziazione neuronale NSC verso un<html> nd lignaggi gliali. Entrambi questi segni transitoriamente si accumulano in concomitanza con aspetto degli indicatori di neuronale precoce e la differenziazione gliale 20. Per determinare la distribuzione delle 5caC nel differenziare NSC abbiamo effettuato co-marcatura di questo segno con un pennarello GFAP gliale sulle culture fissi di NSC a 3 giorni di induzione della differenziazione gliale. A differenza di NSC o astrociti maturi (dati non riportati), abbiamo osservato segnale forte 5caC in percentuale relativamente alta delle cellule che esprimono GFAP in queste culture (Figura 2) 20. <img alt="Figura 1" src="/files/ftp_upload/54416/54416fig1.jpg" /> Figura 1. La co-immunocolorazione di 5hmC (verde) con NeuN (rosso) nel tessuto del cervello adulto di contrasto con DAPI (blu). I singoli canali e la vista dalla fusione sono mostrati. Barre di scala sono 25 micron.blank &quot;&gt; Clicca qui per vedere una versione più grande di questa figura. <img alt="figura 2" src="/files/ftp_upload/54416/54416fig2.jpg" /> Figura 2. Co-immunocolorazione di 5caC con GFAP nella cultura di NSC al Day 3 del Glial Differenziazione. I singoli canali e la vista dalla fusione sono mostrati. Barre di scala sono 20 micron. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54416/54416fig2large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</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.

Il rilevamento di forme modificate di Citosina Uso Sensitive immunoistochimica

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

detection-modified-forms-cytosine-using-sensitive

Nanyang Technological University

Research

JoVE Journal

Biology

7.2K Views.  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.

Sezionamento sottile fetta di preparativi per immunoistochimica

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

Ricerca

Biologia

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.

Prevenire la diffusione della malaria e la dengue utilizzo Zanzare geneticamente modificate

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.

Immunoistochimica: sezioni di paraffina Utilizzando il Vectastain ABC Kit Labs Vector

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.

Immunoistochimica su sezioni di paraffina di epidermide mouse utilizzando anticorpi fluorescenti

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 larvali NMJ Immunoistochimica

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.

La registrazione ottica di attività elettrica in Guinea-maiale Reti enterici utilizzando tensione sensibile Coloranti

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 Addome Immunoistochimica

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 è un metodo sensibile e rapido per il rilevamento del DNA Cytomegaloviral in fissato in formalina, incluse in paraffina biopsia

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.

Colorimetrico rilevamento di batteri Uso tornasole 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. ...

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.