Les travaux pris en charge par des subventions du NIH / NIDCD et la Fondation Schmitt à RP.

Ce protocole a été développé et affiné basé sur le standard radioactifs et non radioactifs des méthodes d&#39;hybridation in situ, développée précédemment par nous et les autres afin de détecter une ou deux espèces transcription dans 1-7 tissu cérébral. Le protocole décrit ci-dessous a une longueur totale de 2 ou 3 jours, selon le nombre d&#39;interruptions de procédure choisie par l&#39;utilisateur final. Toutes les étapes détaillées ci-dessous doivent être menées à température ambiante, à l&#39;exception de l&#39;étape d&#39;hybridation ribosonde et post-hybridation lavages. Les solutions et les tampons requis pour toutes les étapes de cette méthode peut être trouvée à la fin de ce protocole. 1. Préparation des tissus et de sectionnement <ol><li> Décapiter et extrait de cerveau du sujet rapidement, et le placer dans un moule en plastique de taille suffisante. </li><li> Couverture du cerveau avec Tissue-Tek milieu d&#39;inclusion et de placer rapidement le moule en plastique dans un bain dry-ice/alcohol pour congélation rapide. Tissus congelés peuvent être conservés à -80 ° C jusqu&#39;à utilisation. </li><li> L&#39;utilisation d&#39;un cryostat, de recueillir 2 ou 3 coupes de cerveau par glisser sur Superfrost demandé, majoré des diapositives. Epaisseur des sections devrait être de 10 à 12 um. Les diapositives peuvent être conservés à -80 ° C jusqu&#39;à utilisation. </li></ol> 2. Préparation des colonnes Sephadex G50 pour la purification de la sonde «Colonnes Sephadex peuvent être achetés auprès de sources commerciales, cependant, nous fournissons ci-dessous une alternative à faible coût pour la génération de colonnes, ce qui sera nécessaire pour la purification de la sonde. <ol><li> Hydratez quantité adéquate de Sephadex G50 poudre avec RNase, traitée par DEPC eau (par exemple, 2 g de poudre dans 100 ml d&#39;eau traitée au DEPC), brièvement solution de mélanger et conserver à température ambiante pour précipiter l&#39;excès de Sephadex G50. </li><li> Retirer le surnageant (couche supérieure de l&#39;eau) à la suite de Sephadex G50 précipitations. </li><li> Répétez le processus ci-dessus 3 - 5 fois. </li><li> Après le lavage final, re-suspendre le Sephadex G50 solution dans un tampon TE (1:1), et conserver à 4 ° C jusqu&#39;à utilisation. </li><li> Placez autoclavé laine de verre dans une seringue de 1 ml stériles et le compresser avec le piston pour faire une couche compacte au fond, puis placez la seringue dans un tube Falcon de 15 ml. </li><li> Mélangez bien la solution de Sephadex G50 dans TE. Remplir la seringue / colonne avec cette solution. </li><li> Centrifuger la colonne pendant 30 sec à 1000 rpm. </li><li> Répétez cette procédure jusqu&#39;à ce que la colonne est presque complètement rempli de billes de Sephadex G50. </li><li> Appliquer 200 pi de tampon de colonne de lavage à la colonne et on centrifuge pendant 2 min à 1000 rpm. Jeter l&#39;éluat. </li><li> Appliquer 200 pi de tampon de colonne de blocage à la colonne et la faire tourner pendant 2 min à 1000 rpm. Répétez cette étape fois 4-5 à équilibrer la colonne. Les colonnes peuvent être conservés à 4 ° C jusqu&#39;à leur utilisation s&#39;ils sont scellés avec du parafilm. </li></ol> 3. Étiquetage et purification de ribosondes Ci-dessous nous détaillons la production et la purification d&#39;une ribosonde unique. Pour dFISH, la préparation de chaque sonde impliquera la même méthodologie, sauf que l&#39;une des sondes sera marquée à la digoxigénine (DIG)-UTP marqués tandis que l&#39;autre avec de la biotine-UTP marqués. <ol><li> Préparer une solution concentrée (&gt; 150 ng / pl) et solution purifiée d&#39;ADNc linéarisées d&#39;intérêt pour générer ou l&#39;autre sens (de contrôle) ou ribosondes antisens. </li><li> Dans un tube de 1,5 ml, ajouter 0,5 à 1 mg de protéine purifiée de matrice d&#39;ADNc, 2 pl de tampon de la sonde étiquetage 5X, 1 pl de DIG 10X (ou biotine)-étiquetage mélanger, 0,5 pi de RNasine et 1 pl de la ARN polymerase appropriée , et porter le volume final de la solution à 10 ul avec RNAse free (traité au DEPC) d&#39;eau. </li><li> Incuber la solution dans un bain d&#39;eau à 37 ° C pendant 2 heures. </li><li> Ajouter 1 ul d&#39;ARNt (stock; 20 pg / pl), et 39 pi de tampon de colonne de blocage à la solution. </li><li> Préparer la colonne de Sephadex G50 pour la purification de la sonde. À cette fin, ajouter 50 ul de tampon de blocage à la colonne et la faire tourner pendant 10 sec à 1000 rpm. Répétez cette étape 2-3 fois pour équilibrer la colonne (ie, le 50 ul appliquée à la colonne sont récupérés après cycle de centrifugation). </li><li> Appliquer la solution de sonde à l&#39;Sephadex G50 colonne, la position d&#39;un tube de centrifugeuse nouvelles au bas de la colonne, et la faire tourner pendant 3 min à 1000 rpm pour obtenir le purifiée 50 ul de solution ribosonde. </li><li> Évaluer la qualité et le rendement de ribosonde marquée en utilisant soit une norme de formaldéhyde-agarose ARN gel, ou un spectrophotomètre. </li></ol> 4. Post-fixation, l&#39;acétylation et hybridation <ol><li> Supprimez les parties du congélateur à -80 ° C et permettent une température ambiante de s&#39;équilibrer. Par la suite, incuber sections dans un froid, fraîchement fait une solution de paraformaldéhyde 3% pendant 5 min. </li><li> Rincer brièvement sections phosphate saline tamponnée (PBS) à deux reprises. </li><li> Déshydrater sections à travers une série d&#39;alcool type (70, 95 et 100%; 2 minutes chacun), et laissez-les sécher à l&#39;air. </li><li> Incuber sections dans une solution acétylation pendant 10 min. </li><li> Rincer les sections 3 fois dans 2X SSPE. </li><li> Déshydrater sections une fois de plus grâce à la série d&#39;alcool standard décrit ci-dessus, et leur permettre de sécher à l&#39;air. </li><li> Préparer un volume adéquat de solution d&#39;hybridation et d&#39;ajouter deux ribosondes à la solution (concentration de la sonde: 1 ng / pl pour chaque sonde). Le volume total de la solution d&#39;hybridation est déterminée en fonction du nombre de sections à hybrider (16 ul de solution d&#39;hybridation par section). </li><li> Appliquer un volume suffisant de la solution d&#39;hybridation des lames de tissu et la lamelle veiller à ce qu&#39;aucun des bulles d&#39;air superposition des tissus. </li><li> Placer les lames dans un support de diapositives de métal. Titulaire Plongez dans un bain d&#39;huile minérale réglé à 65 ° C pendant la nuit. Assurez-vous que des lamelles sont face vers le haut (c&#39;est à dire, la face latérale du support de diapositives doivent être en contact avec le fond du récipient d&#39;huile minérale). </li></ol> 5. Post-hybridation Lave <ol><li> Le lendemain, retirez soigneusement le support de diapositives du bain d&#39;huile minérale et brièvement le rincer dans du chloroforme pour éliminer l&#39;excès d&#39;huile à partir des diapositives. </li><li> Placer les lames dans une solution de 2X SSPE pour les 5-10 min. Lamelles doit se détacher de diapositives tout en solution. </li><li> Placer les lames dans une nouvelle solution 2X SSPE, et de les garder pendant 1 heure à température ambiante. </li><li> Transfert des sections dans une solution contenant 2X SSPE, plus 50% de formamide. La température de cette solution doit correspondre à la température utilisée dans la procédure de l&#39;hybridation pendant la nuit. Gardez diapositives dans cette solution pendant 1,5 heures. </li><li> Transfert des sections d&#39;une solution 0,1 X SSPE préchauffée à la même température de l&#39;étape d&#39;hybridation. Incuber sections dans cette solution pendant 30 min. Répétez cette étape pour 30 minutes supplémentaires. </li></ol> 6. Détection et visualisation de ribosondes <ol><li> Transfert diapositives tampon TNT à laquelle le peroxyde d&#39;hydrogène de 0,3% a été ajoutée, pour 10 min. </li><li> Lavez sections dans du tampon TNT, 3 fois (10 minutes chacun). </li><li> Utiliser un stylo DAKO, dessiner un puits autour de la zone contenant les coupes de cerveau. Surtout, il faut être prudent afin d&#39;assurer que les articles ne se dessèchent pas. </li><li> Appliquer 150 pi de tampon TNB à chaque diapositive, et incuber sections dans cette solution pendant 30 min, dans une chambre humide. </li><li> Retirer l&#39;excès solution TNB par des diapositives basculement. </li><li> Appliquer 150 ul de solution contenant TNB-peroxydase anticorps anti-DIG, et glisse stocker dans une chambre humide pendant 2 heures. Concentration d&#39;anticorps doit être déterminée individuellement pour chaque transcription d&#39;intérêt avant d&#39;effectuer dFISH. </li><li> Lavez sections dans du tampon TNT; 3 fois, 10 minutes chacun. </li><li> Appliquer 150 ul d&#39;Alexa 594-conjugué solution tyramide travail pour chaque diapositive, et les stocker dans une chambre humide pendant 1 heure. Cette solution doit être préparée selon les instructions du fabricant. </li><li> Lavez sections dans du tampon TNT; 3 fois, 10 minutes chacun. </li><li> Sections Incuber dans tampon TNT à laquelle le peroxyde d&#39;hydrogène de 0,3% a été ajoutée pour 10 - 30 min. </li><li> Lavez sections dans du tampon TNT; 3 fois, 10 minutes chacun. </li><li> Appliquer 150 pi de tampon TNB par diapositive, et les garder dans une chambre humide pendant 30 min. </li><li> Retirer l&#39;excès solution TNB par des diapositives basculement. </li><li> Ajouter 150 ul de la solution contenant TNB conjugué à la peroxydase d&#39;anticorps anti-biotine, et glisse stocker dans une chambre humide pendant 2 heures. </li><li> Lavez sections dans du tampon TNT; 3 fois, 10 minutes chacun. </li><li> Appliquer 150 ul d&#39;un Alexa 488-conjugué solution tyramide travail pour les diapositives et les sections incuber pendant 1 heure. Cette solution doit être préparée conformément aux recommandations du fabricant. </li><li> Lavez sections dans du tampon TNT; 3 fois, 10 minutes chacun. </li><li> Ajouter 150 pi de solution de Hoechst (1:1000 dans du tampon TNT) pour les sections et les garder dans la chambre humide pendant 2 min. </li><li> Laver les coupes dans du tampon TNT; 3 fois, 5 minutes chacun. </li><li> Sections lamelle avec un milieu de montage compatibles fluorescence (par exemple, ou de prolonger Vectashield Antifade). </li></ol> 7. Les résultats représentatifs <img src="/files/ftp_upload/2102/2102fig1.jpg" alt="Figure 1" /> Figure 1. Nous montrons ici les résultats obtenus dFISH représentatives dans le cerveau diamant mandarin. Illustré sont des microphotographies obtenues à partir des caudomedial nidopallium (MR), l&#39;analogue d&#39;oiseaux chanteurs du cortex auditif des mammifères. Coupes de cerveau ont été hybridés avec une ribosonde biotinylé dirigé contre la parvalbumine (A), un marqueur d&#39;une sous-population de neurones inhibiteurs, et une DIG marqué ribola sonde dirigée contre les Zenk dépendant de l&#39;activité du gène (B), un marqueur fiable de la chanson axé sur les neurones. C) Superposition de (A) et (B) montre une population de neurones inhibiteurs qui sont activés par l&#39;expérience auditive. Flèches et pointes de flèches indiquent les cellules marquées exclusivement avec chacun des deux ribosondes, et les astérisques indiquent les neurones représentant de co-exprimant les deux transcriptions d&#39;intérêt. Barre d&#39;échelle = 25 um.

<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>

Nous avons utilisé ce protocole pour étudier comment le cerveau des vertébrés est neurochemically et fonctionnellement organisés, et de déterminer comment behaviorally pertinentes impact sensoriel des stimuli de la génomique des machines de neurones dans le cerveau adulte de 80 à 10. Nous avons utilisé avec succès cette méthode dans le tissu cérébral de souris, les rats et les oiseaux chanteurs, mais attendons à ce que ce protocole sera facilement adaptable à des sections de cerveau obtenue à ...

Solutions <ol><li> Tampon de colonne de lavage: 10 mM Tris-HCl, 0,15 M de NaCl, 0,05 mM EDTA, 50 ug / ul ARNt, SDS 0,1% dans 50 ml d&#39;eau traitée par DEPC. </li><li> Tampon de colonne de blocage: 10 mM Tris-HCl, 50 mM de NaCl, 0,1 mM d&#39;EDTA dans 50 ml d&#39;eau traitée par DEPC. </li><li> Tampon TNT: 60 ml de 1 M Tris-HCl, 18 ml de NaCl 5 M et 1,8 ml de Triton X-100 dans 600 ml d&#39;eau traitée au DEPC. </li><li> TNB mémoire tampon: 100 mM Tris-HCl, 8,3 g / ul BSA, 0,15 M de NaCl et 3% de Triton X-100 dans l&#39;eau traitée au DEPC. </li><li> Tampon TE: 10 mM Tris-HCI, pH 7,5 et 1 mM EDTA, pH 7,5. </li><li> L&#39;acétylation solution: 2,7 ml de triéthanolamine majoré de 0,5 ml d&#39;anhydride acétique dans 200 ml d&#39;eau traitée au DEPC. </li><li> La solution d&#39;hybridation: 50% de formamide, 2X SSPE, 2 ug / ul ARNt, 1 ug / ul BSA et 1 ug / ul poly A dans l&#39;eau traitée par DEPC. Il a besoin de 16 l par article. </li></ol>

double fluorescence in situ hybridization in fresh brain sections

Nous décrivons ici une version modifiée d&#39;une fluorescence à double<em&gt; In situ</emL&#39;hybridation&gt; (dFISH) méthode optimisée pour la détection de deux ARNm d&#39;intérêt dans des coupes de cerveau frais congelé. Notre groupe a utilisé avec succès cette approche pour étudier des gènes co-régulation. Plus précisément, nous avons utilisé cette méthode pour explorer dFISH l&#39;organisation anatomique, les propriétés neurochimiques, et l&#39;impact de l&#39;expérience sensorielle dans le centre de circuits sensoriels, à la résolution seule cellule. Ce protocole a été validé dans le tissu cérébral de souris, les rats et les oiseaux chanteurs, mais devrait être facilement adaptable à d&#39;autres espèces de vertébrés, ainsi que d&#39;un éventail de tissus non neuraux. Dans ce film nous fournir une démonstration détaillée des principales étapes de cette procédure.

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

Ce protocole implique un non-radioactifs<em&gt; In-situ</emProcessus d&#39;hybridation&gt; qui permet l&#39;identification simultanée des deux espèces transcription, à une résolution seule cellule, dans les sections minces du cerveau des vertébrés.

Double fluorescence In situ</emHybridation> dans des coupes de cerveau frais

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

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Neuroscience

Double Fluorescence in situ Hybridization in Fresh Brain Sections

16.0K vues.  University of Rochester. Ce protocole implique un non-radioactifs In-situ

Vidéo: 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.

Combinant colorant lipophile, In situ</emHybridation>, immunohistochimie et l&#39;histologie

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

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Neurosciences

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

Conception et montage d&#39;un Microdrive ultra-léger motorisé pour des enregistrements de neurones chroniques chez les petits animaux

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

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.

Spinal Cord Injury complète et cerveau Protocole Dissection pour Wholemount suite<em&gt; In Situ</em&gt; Hybridation dans des larves de la lamproie marine

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.

Détection de axonaux localisée ARNm dans les sections de cerveau en utilisant haute résolution<em&gt; In Situ</em&gt; Hybridation

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

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.

Rapid<em&gt; In Situ</em&gt; Hybridation utilisant sondes oligonucléotidiques sur Brain Paraformaldéhyde préfixée de rats avec syndrome sérotoninergique

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.

Combinant Double Fluorescence<em&gt; In Situ</em&gt; Hybridation avec Immunomarquage pour la détection de l&#39;expression de trois gènes chez la souris Sections du cerveau

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

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.

Localisation des gènes des récepteurs odorants dans les antennes acridiennes par ARN<em&gt; In Situ</em&gt; Hybridation

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.

Combinaison de l’hybridation in situ par fluorescence multiplex avec l’immunohistochimie fluorescente sur des coupes de cerveau de souris fraîches, congelées ou fixes

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 modèle de rat d’infection cérébrale EcoHIV

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

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

Double fluorescence In situ dans des coupes de cerveau frais