Lavoro sostenuto dalle concessioni di NIH / dell&#39;NIDCD e la Fondazione Schmitt a RP.

Questo protocollo è stato sviluppato e raffinato basato sullo standard radioattivi e non radioattivi in situ metodi di ibridazione, in precedenza sviluppato da noi e gli altri di individuare uno o due specie di trascrizione nel tessuto cerebrale 1-7. Il protocollo descritto di seguito ha una lunghezza totale di 2 o 3 giorni, a seconda del numero di interruzioni procedurale scelto dall&#39;utente finale. Tutti i passaggi dettagliati di seguito sono essere condotte a temperatura ambiente, con l&#39;eccezione della fase di ibridazione riboprobe e post-ibridazione lavaggi. Le soluzioni e buffer richiesto per tutti i passi necessari per questo metodo si possono trovare alla fine di questo protocollo. 1. Preparazione dei tessuti e di sezionamento <ol><li> Decapitare ed estrarre il cervello del soggetto rapidamente, e mettetelo in uno stampo di plastica di dimensioni adeguate. </li><li> Coprire con tessuto cerebrale-Tek mezzo di inclusione e rapidamente posto lo stampo di plastica in un bagno dry-ice/alcohol per il congelamento rapido. Tessuti congelati possono essere conservati a -80 ° C fino al momento dell&#39;uso. </li><li> Utilizzando un criostato, raccogliere 2 o 3 sezioni di cervello per vetrino sul Superfrost carica Plus. diapositive. Spessore delle sezioni dovrebbe essere 10-12 micron. Le diapositive possono essere conservati a -80 ° C fino al momento dell&#39;uso. </li></ol> 2. Preparazione di Sephadex G50 colonne per purificazione della sonda &quot;Colonne Sephadex possono essere acquistati da fonti commerciali, però, mettiamo a disposizione di sotto di una alternativa a basso costo per la generazione di colonne, che sarà necessario per la purificazione della sonda. <ol><li> Idrato quantità adeguata di Sephadex G50 polvere con RNase-free, DEPC trattati con acqua (per esempio, 2 g di polvere in 100 ml di DEPC trattati con acqua), soluzione brevemente mescolare e conservare a temperatura ambiente per far precipitare l&#39;eccesso Sephadex G50. </li><li> Rimuovere il surnatante (strato superiore di acqua) a seguito Sephadex G50 precipitazioni. </li><li> Ripetere la procedura sopra 3 - 5 volte. </li><li> Dopo l&#39;ultimo lavaggio, risospendere la soluzione G50 Sephadex in TE buffer (rapporto 1:1), e conservare a 4 ° C fino all&#39;uso. </li><li> Luogo in autoclave lana di vetro in una sterile siringa da 1 ml e comprimerlo con lo stantuffo di fare uno strato compatto sul fondo, quindi posizionare la siringa in una provetta da 15 ml Falcon. </li><li> Mescolare bene la soluzione di Sephadex G50 a TE. Riempire la siringa / colonna con questa soluzione. </li><li> Centrifugare la colonna per 30 sec a 1000 giri. </li><li> Ripetere questa procedura fino a quando la colonna è quasi completamente riempito di Sephadex G50 perline. </li><li> Applicare 200 l di tampone colonna di lavaggio alla colonna e centrifugare per 2 min a 1000 rpm. Gettare flow-through. </li><li> Applicare 200 l di tampone colonna blocco alla colonna e centrifugare per 2 min a 1000 rpm. Ripetere questo passo 4-5 volte per equilibrare colonna. Le colonne possono essere conservati a 4 ° C fino all&#39;utilizzo se sigillati con parafilm. </li></ol> 3. L&#39;etichettatura e la Purificazione di Riboprobes Di seguito dettaglio la generazione e la purificazione di un singolo riboprobe. Per dFISH, la preparazione di ciascuna sonda coinvolgerà la stessa metodologia, salvo che una delle due sonde sarà marcato con digossigenina (DIG)-tagged UTP, mentre l&#39;altro con biotina-tagged UTP. <ol><li> Preparare un concentrato (&gt; 150 ng / mL) e purificate soluzione linearizzata cDNA di interesse sia per generare senso (controllo) o riboprobes antisenso. </li><li> In una provetta da microcentrifuga 1,5 ml, aggiungere 0,5-1 mg di cDNA purificato modello, 2 ml di tampone 5X etichettatura sonda, 1 l di DIG 10X (o biotina)-etichettatura mix, 0,5 l di RNasin e 1 microlitri della polimerasi RNA appropriato , e portare il volume finale della soluzione a 10 microlitri con RNasi-free (DEPC-trattati) l&#39;acqua. </li><li> Incubare la soluzione in un bagno d&#39;acqua a 37 ° C per 2 ore. </li><li> Aggiungere 1 ml di tRNA (magazzino; 20 mg / mL), e 39 ml di buffer di colonna di blocco per la soluzione. </li><li> Preparare il Sephadex G50 colonna per la purificazione della sonda. A tal fine, aggiungere 50 microlitri di buffer di blocco per la colonna e girare per 10 secondi a 1000 giri. Ripetere questa operazione 2-3 volte per equilibrare la colonna (cioè, il pieno 50 microlitri applicata alla colonna vengono recuperati dopo il ciclo di centrifugazione). </li><li> Applicare la soluzione della sonda alla colonna Sephadex G50, la posizione di una provetta per microcentrifuga nuovo sul fondo della colonna, e girare per 3 min a 1000 rpm per ottenere il purificata 50 ml di soluzione riboprobe. </li><li> Valutare la qualità e la resa di riboprobe etichettati utilizzando uno standard di formaldeide-agarosio RNA gel o uno spettrofotometro. </li></ol> 4. Post-fissazione, acetilazione e ibridazione <ol><li> Rimuovere sezioni dal freezer ° -80 C e permettere una temperatura ambiente per equilibrare. Successivamente, incubare sezioni di un freddo, appena reso soluzione paraformaldeide al 3% per 5 min. </li><li> Brevemente risciacquare sezioni in phosphate soluzione salina tamponata (PBS) due volte. </li><li> Disidratare sezioni attraverso una serie standard di alcool (70, 95, e 100%, 2 minuti ciascuna), e lasciarli asciugare all&#39;aria. </li><li> Incubare le sezioni in una soluzione acetilazione per 10 minuti. </li><li> Sciacquare le sezioni 3 volte in 2X SSPE. </li><li> Disidratare sezioni ancora una volta attraverso la serie di alcol standard descritto in precedenza, e permettere loro di asciugare all&#39;aria. </li><li> Preparare un adeguato volume di soluzione di ibridazione e aggiungere entrambi riboprobes alla soluzione (concentrazione di sonde: 1 ng / mL per ogni sonda). Volume totale della soluzione di ibridazione è determinato in base al numero di sezioni da ibridato (16 ml di soluzione di ibridazione per sezione). </li><li> Applicare un adeguato volume della soluzione di ibridazione alle diapositive tessuti e coprioggetto assicurando che nessun sovrapposizione bolle d&#39;aria il tessuto. </li><li> Posizionare i vetrini in un portavetrini metallo. Titolare immergere in un bagno di olio minerale fissata a 65 ° C durante la notte. Assicurarsi che coprioggetto sono rivolto verso l&#39;alto (cioè, l&#39;aspetto laterale del supporto per diapositive devono essere a contatto con il fondo del contenitore olio minerale). </li></ol> 5. Post-ibridazione lava <ol><li> Il giorno seguente, rimuovere con attenzione il supporto diapositive dal bagno d&#39;olio minerale e brevemente sciacquarlo in cloroformio per rimuovere l&#39;olio in eccesso dal diapositive. </li><li> Posizionare i vetrini in una soluzione di 2X SSPE per 5-10 min. Coprioggetto dovrebbe staccarsi da diapositive, mentre in soluzione. </li><li> Trasferire i vetrini in una nuova soluzione 2X SSPE, e tenerli per 1 ora a temperatura ambiente. </li><li> Trasferire le sezioni in una soluzione contenente 2X SSPE più 50% formammide. La temperatura di questa soluzione dovrebbe corrispondere alla temperatura utilizzata nella procedura di ibridazione durante la notte. Tenere diapositive in questa soluzione per 1,5 ore. </li><li> Trasferimento sezioni per una soluzione di 0,1 X. SSPE preriscaldata alla stessa temperatura della fase di ibridazione. Incubare sezioni in questa soluzione per 30 min. Ripetere questo passaggio per altri 30 min. </li></ol> 6. Rilevazione e visualizzazione di Riboprobes <ol><li> Trasferimento diapositive a tampone TNT a cui perossido di idrogeno 0,3% è stato aggiunto, per 10 minuti. </li><li> Lavare le sezioni in TNT tampone, 3 volte (10 min ciascuna). </li><li> Utilizzando una penna DAKO, disegnare un bene intorno l&#39;area che contiene le sezioni del cervello. Soprattutto, si deve usare cautela per garantire che le sezioni non si asciugano. </li><li> Applicare 150 ml di tampone TNB di ogni diapositiva, e incubare sezioni in questa soluzione per 30 minuti, in una camera umida. </li><li> Rimuovere la soluzione in eccesso TNB da diapositive inclinazione. </li><li> Applicare 150 ml di soluzione contenente TNB perossidasi-coniugato anticorpo anti-DIG, e conservare i vetrini in camera umida per 2 ore. Concentrazione anticorpale deve essere determinata individualmente per ogni trascrizione di interesse prima di effettuare dFISH. </li><li> Lavare le sezioni in TNT buffer; 3 volte, ogni 10 min. </li><li> Applicare 150 ml di Alexa 594-coniugato soluzione tyramide lavorando per ogni diapositiva, e memorizzarli in una camera umida per 1 ora. Questa soluzione deve essere preparata secondo le istruzioni del produttore. </li><li> Lavare le sezioni in TNT buffer; 3 volte, ogni 10 min. </li><li> Sezioni incubare nel buffer TNT a cui perossido di idrogeno 0,3% è stata aggiunta per 10 - 30 min. </li><li> Lavare le sezioni in TNT buffer; 3 volte, ogni 10 min. </li><li> Applicare 150 ml di tampone TNB per vetrino, e tenerli in camera umida per 30 min. </li><li> Rimuovere la soluzione in eccesso TNB da diapositive inclinazione. </li><li> Aggiungere 150 ml di soluzione contenente TNB perossidasi-coniugato anticorpi anti-biotina, e conservare i vetrini in camera umida per 2 ore. </li><li> Lavare le sezioni in TNT buffer; 3 volte, ogni 10 min. </li><li> Applicare 150 ml di uno Alexa 488-coniugato soluzione tyramide al lavoro per le diapositive e le sezioni incubare per 1 ora. Questa soluzione deve essere preparata secondo le raccomandazioni del produttore. </li><li> Lavare le sezioni in TNT buffer; 3 volte, ogni 10 min. </li><li> Aggiungere 150 ml di soluzione Hoechst (1:1000 in TNT buffer) per le sezioni e tenerli in camera umida per 2 minuti. </li><li> Lavare le sezioni in TNT buffer; 3 volte, 5 minuti ciascuno. </li><li> Sezioni coprioggetto con una fluorescenza compatibile mezzo di montaggio (ad esempio, Vectashield o prolungare Antifade). </li></ol> 7. Rappresentante Risultati <img src="/files/ftp_upload/2102/2102fig1.jpg" alt="Figura 1" /> Figura 1. Mostriamo qui i risultati ottenuti dFISH rappresentante nel cervello diamante mandarino. Vengono mostrati microfotografie ottenuti dal caudomedial nidopallium (NCM), l&#39;analogo songbird della corteccia uditiva dei mammiferi. Sezioni di cervello sono stati ibridati con un riboprobe biotinilato diretta contro parvalbumina (A), un marker per una sub-popolazione di neuroni inibitori, e una DIG marcato ribosonde dirette contro l&#39;attività del gene-dipendente Zenk (B), un marker affidabile per la canzone-driven neuroni. C) Sovrapposizione di (A) e (B) mostra una popolazione di neuroni inibitori che sono attivati ​​attraverso l&#39;esperienza uditiva. Frecce e frecce indicano le cellule etichettate esclusivamente con ciascuno dei due riboprobes, e asterischi mostrano neuroni rappresentante co-esprimono entrambi trascrizioni di interesse. Barra di scala = 25 micron.

<ol>
	<li>Jin, L., Lloyd, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization:+methods+and+applications.">In situ hybridization: methods and applications.</a> J Clin Lab Anal. 11, 2-9 (1997).</li><li>Stoler, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization.">In situ hybridization.</a> Clin Lab Med. 10, 215-236 (1990).</li><li>Komminoth, P., Werner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Target+and+signal+amplification:+approaches+to+increase+the+sensitivity+of+in+situ+hybridization.">Target and signal amplification: approaches to increase the sensitivity of in situ hybridization.</a> Histochem Cell Biol. 108, 325-3233 (1997).</li><li>Kessler, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+digoxigenin:anti-digoxigenin+(DIG)+technology--a+survey+on+the+concept+and+realization+of+a+novel+bioanalytical+indicator+system.">The digoxigenin:anti-digoxigenin (DIG) technology--a survey on the concept and realization of a novel bioanalytical indicator system.</a> Mol Cell Probes. 5, 161-205 (1991).</li><li>Panoskaltsis-Mortari, A., Bucy, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+hybridization+with+digoxigenin-labeled+RNA+probes:+facts+and+artifacts.">In situ hybridization with digoxigenin-labeled RNA probes: facts and artifacts.</a> Biotechniques. 18, 300-307 (1995).</li><li>Qian, X., Lloyd, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+developments+in+signal+amplification+methods+for+in+situ+hybridization.">Recent developments in signal amplification methods for in situ hybridization.</a> Diagn Mol Pathol. 12, 1-13 (2003).</li><li>Mello, C. V., Jarvis, E. D., Denisenko, N., Rivas, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+song-regulated+genes+in+the+brain+of+songbirds.">Isolation of song-regulated genes in the brain of songbirds.</a> Methods Mol Biol. 85, 205-217 (1997).</li><li>Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+neurons+participate+in+the+brain's+response+to+birdsong+auditory+stimulation.">GABAergic neurons participate in the brain's response to birdsong auditory stimulation.</a> Eur J Neurosci. 20, 1318-1330 (2004).</li><li>Velho, T. A., Pinaud, R., Rodrigues, P. V., Mello, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-induction+of+activity-dependent+genes+in+songbirds.">Co-induction of activity-dependent genes in songbirds.</a> Eur J Neurosci. 22, 1667-1678 (2005).</li><li>Tremere, L. A., Jeong, J. K., Pinaud, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estradiol+shapes+auditory+processing+in+the+adult+brain+by+regulating+inhibitory+transmission+and+plasticity-associated+gene+expression.">Estradiol shapes auditory processing in the adult brain by regulating inhibitory transmission and plasticity-associated gene expression.</a> J Neurosci. 29, 5949-5963 (2009).</li><li>Pinaud, R., Mello, C. V., Velho, T. A., Wynne, R. D., Tremere, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+two+mRNA+species+at+single-cell+resolution+by+double-fluorescence+in+situ+hybridization.">Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.</a> Nat Protoc. 3, 1370-1379 (2008).</li><li>Pinaud, R., Jeong, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplex+fluorescence+in+situ+hybridization+in+the+study+of+gene+co-regulation+in+the+vertebrate+brain.+Methods+Mol+Biol.">Duplex fluorescence in situ hybridization in the study of gene co-regulation in the vertebrate brain. Methods Mol Biol.</a> 611, 115-129 (2010).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo usato questo protocollo per studiare come il cervello dei vertebrati è neurochemically e funzionalmente organizzato, e per determinare come comportamentale rilevante per l&#39;impatto sensoriale stimoli il meccanismo genomico dei neuroni nel cervello adulto 8-10. Abbiamo utilizzato con successo questo metodo nel tessuto cerebrale di topi, ratti e uccelli canori, ma prevediamo che questo protocollo sarà facilmente adattabile alle sezioni del cervello ottenute da una serie di specie di vertebrati e, f...

Soluzioni <ol><li> Colonna del buffer di lavaggio: 10 mM Tris-HCl 0,15 M NaCl, 0,05 mM EDTA, 50 mg / tRNA microlitri, 0,1% SDS in 50 ml DEPC-acqua trattata. </li><li> Colonna tampone di bloccaggio: 10 mM Tris-HCl, 50 mM NaCl, 0,1 mM EDTA in 50 ml DEPC-acqua trattata. </li><li> TNT buffer: 60 ml di 1 M Tris-HCl, 18 ml di NaCl 5 M e 1,8 ml di Triton X-100 in 600 ml di DEPC trattati con acqua. </li><li> TNB buffer: 100 mM Tris-HCl, 8.3 mg / microlitri BSA, 0,15 M di NaCl e il 3% Triton X-100 in DEPC trattati con acqua. </li><li> TE buffer: 10 mM Tris-HCl, pH 7,5 e 1 mM EDTA, pH 7,5. </li><li> Acetilazione soluzione: 2,7 ml di trietanolammina più 0,5 ml di anidride acetica in 200 ml di DEPC trattati con acqua. </li><li> Ibridazione soluzione: il 50% formammide, 2X SSPE, 2 mg / tRNA microlitri, 1 mg / BSA microlitri e 1 mg / mL poli A in acqua DEPC-trattata. Ha bisogno di 16 l per sezione. </li></ol>

double fluorescence in situ hybridization in fresh brain sections

Qui si descrive una versione modificata di una fluorescenza doppio<em&gt; In situ</emIbridazione&gt; (dFISH) metodo ottimizzato per il rilevamento di due mRNA di interesse fresco sezioni cervello congelato. Il nostro gruppo ha utilizzato con successo questo approccio per lo studio genetico di co-regolamentazione. Più in particolare, abbiamo utilizzato questo metodo dFISH per esplorare l&#39;organizzazione anatomica, le proprietà neurochimiche, e l&#39;impatto dell&#39;esperienza sensoriale nel centro di circuiti sensoriali, a risoluzione singola cellula. Questo protocollo è stato convalidato nel tessuto cerebrale di topi, ratti e uccelli canori, ma dovrebbe essere facilmente adattabile alle altre specie di vertebrati, come pure una serie di non-tessuti neurali. In questo film ci fornisce una dimostrazione dettagliata delle tappe principali di questa procedura.

Watch this Scientific Journal Video about Double Fluorescence in situ Hybridization in Fresh Brain Sections at JoVE.com

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Questo protocollo comporta un non radioattivo<em&gt; In situ</emIbridazione&gt; procedura che consente l&#39;identificazione simultanea di due specie di trascrizione, ad una risoluzione singola cella, in sezioni sottili del cervello dei vertebrati.

Doppia fluorescenza In situ</emIbridazione> nelle sezioni cervello fresco

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

double-fluorescence-in-situ-hybridization-in-fresh-brain-sections

Research

JoVE Journal

Neuroscience

Double Fluorescence in situ Hybridization in Fresh Brain Sections

15.9K Views.  University of Rochester. Questo protocollo comporta un non radioattivo In situ procedura che consente l'identificazione simultanea di due specie di trascrizione, ad una risoluzione singola cella, in sezioni sottili del cervello dei vertebrati.

Video: Double Fluorescence in situ Hybridization in Fresh Brain Sections

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

La combinazione di coloranti lipofili, In situ Ibridazione, Immunoistochimica e Istologia

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Ricerca

Neuroscienze

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

Completa lesioni del midollo spinale e del cervello dissezione Protocollo per Wholemount successiva<em&gt; In Situ</em&gt; Ibridazione in larvale Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

Rilevazione di assoni localizzato mRNA nelle sezioni del cervello Utilizzando ad alta risoluzione<em&gt; In Situ</em&gt; Ibridazione

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Double-barreled and Concentric Microelectrodes for Measurement of Extracellular Ion Signals in Brain Tissue

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular concentration of all major ions. As these ion shifts bear significant functional consequences, their quantitative determination is often required to understand the function and dysfunction of neural networks under physiological and pathophysiological conditions. In the present study, we demonstrate the fabrication and calibration of double-barreled ion-selective microelectrodes, which have proven to be excellent tools for such measurements in brain tissue. Moreover, so-called &#8220;concentric&#8221; ion-selective microelectrodes are also described, which, based on their different design, offer a far better temporal resolution of fast ion changes. We then show how these electrodes can be employed in acute brain slice preparations of the mouse hippocampus. Using double-barreled, potassium-selective microelectrodes, changes in the extracellular potassium concentration ([K+]o) in response to exogenous application of glutamate receptor agonists or during epileptiform activity are demonstrated. Furthermore, we illustrate the response characteristics of sodium-sensitive, double-barreled and concentric electrodes and compare their detection of changes in the extracellular sodium concentration ([Na+]o) evoked by bath or pressure application of drugs. These measurements show that while response amplitudes are similar, the concentric sodium microelectrodes display a superior signal-to-noise ratio and response time as compared to the double-barreled design. Generally, the demonstrated procedures will be easily transferable to measurement of other ions species, including pH or calcium, and will also be applicable to other preparations.

We demonstrate the fabrication, calibration and properties of two types of ion-selective microelectrodes (double-barreled and concentric) for measurement of ion concentrations in brain tissue. These are then used in the mouse hippocampal slice preparation to show that excitatory activity changes both extracellular potassium and sodium concentrations.

Microelettrodi doppia canna e concentrico per la misurazione di extracellulare Ion Segnali in tessuto cerebrale

Electrical activity in the brain is accompanied by significant ion fluxes across membranes, resulting in complex changes in the extracellular ...

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

Rapido<em&gt; In Situ</em&gt; Ibridazione utilizzando oligonucleotidi sonde su Paraformaldeide prefissa cervello di ratti con Sindrome serotoninergica

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

La combinazione di doppio Fluorescenza<em&gt; In Situ</em&gt; Ibridazione con Immunolabelling per il rilevamento della Manifestazione di tre geni nel cervello di topo Sezioni

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

Fluorescence Activated di separazione delle cellule (FACS) e Gene Expression Analisi di Fos-esprimono neuroni da freschi e congelati Rat tessuto cerebrale

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

Localizzazione dei geni del recettore degli odoranti nell&#39;antenna di Locust da RNA<em&gt; In Situ</em&gt; Ibridazione

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within cells. Neurochemical phenotyping of functionally identified neurons usually requires concurrent labelling with multiple antibodies (targeting protein) using immunohistochemistry (IHC) and optimization of in situ hybridization (targeting RNA), in tandem. A &#34;neurochemical signature&#34; to characterize particular neurons may be achieved however complicating factors include the need to verify FISH and IHC targets before combining the methods, and the limited number of RNAs and proteins that may be targeted simultaneously within the same tissue section.
Here we describe a protocol, using both fresh frozen and fixed mouse brain preparations, which detects multiple mRNAs and proteins in the same brain section using RNAscope FISH followed by fluorescence immunostaining, respectively. We use the combined method to describe the expression pattern of low abundance mRNAs (e.g., galanin receptor 1) and high abundance mRNAs (e.g., glycine transporter 2), in immunohistochemically identified brainstem nuclei.
Key considerations for protein labelling downstream of the FISH assay extend beyond tissue preparation and optimization of FISH probe labelling. For example, we found that antibody binding and labelling specificity can be detrimentally affected by the protease step within the FISH probe assay. Proteases catalyze hydrolytic cleavage of peptide bonds, facilitating FISH probe entry into cells, however they may also digest the protein targeted by the subsequent IHC assay, producing off target binding. The subcellular location of the targeted protein is another factor contributing to IHC success following FISH probe assay. We observed IHC specificity to be retained when the targeted protein is membrane bound, whereas IHC targeting cytoplasmic protein required extensive troubleshooting. Finally, we found handling of slide-mounted fixed frozen tissue more challenging than fresh frozen tissue, however IHC quality was overall better with fixed frozen tissue, when combined with RNAscope.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Combinazione dell'ibridazione in situ a fluorescenza multiplex con immunoistochimica fluorescente su sezioni cerebrali di topo fresche, congelate o fisse

Fluorescent in situ hybridization (FISH) is a molecular technique that identifies the presence and spatial distribution of specific RNA transcripts within ...

A Rat Model of EcoHIV Brain Infection

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. Establishment of the EcoHIV infected rat model for studies of drug abuse and neurocognitive disorders, would be beneficial in the study of neuroHIV and HIV-1 associated neurocognitive disorders (HAND). In the present study, we demonstrate the successful creation of a rat model of active HIV infection using chimeric HIV (EcoHIV). First, the lentiviral construct of EcoHIV was packaged in cultured 293 FT cells for 48 hours. Then, the conditional medium was concentrated and titered. Next, we performed bilateral stereotaxic injections of the EcoHIV-EGFP into F344/N rat brain tissue. One week after infection, EGFP fluorescence signals were detected in the infected brain tissue, indicating that EcoHIV successfully induces an active HIV infection in rats. In addition, immunostaining for the microglial cell marker, Iba1, was performed. The results indicated that microglia were the predominant cell type harboring EcoHIV. Furthermore, EcoHIV rats exhibited alterations in temporal processing, a potential underlying neurobehavioral mechanism of HAND as well as synaptic dysfunction eight weeks after infection. Collectively, the present study extends the EcoHIV model of HIV-1 infection to the rat offering a valuable biological system to study HIV-1 viral reservoirs in the brain as well as HAND and associated comorbidities such as drug abuse.

Here, we present a protocol to establish a new rat model of active HIV infection using chimeric HIV (EcoHIV), which is critical for enhancing our understanding of HIV-1 viral reservoirs in the brain and offering a system to study HIV-associated neurocognitive disorders and associated comorbidities (i.e., drug abuse).

Un modello di ratto di infezione cerebrale EcoHIV

It has been well studied that the EcoHIV infected mouse model is of significant utility in investigating HIV associated neurological complications. ...

Doppia fluorescenza In situ nelle sezioni cervello fresco