Nous remercions le Dr Yoko Nabeshima l&#39;Université de Kyoto pour son instruction sur la technique de dissection et Mme Aki Miyakawa au Fujita University Health pour son soutien au cinéma. Ce travail a été soutenu par le Programme pour la promotion des recherches fondamentales en sciences de la santé de l&#39;Institut national de l&#39;innovation biomédicale, une subvention en aide pour la recherche scientifique sur les priorités de recherche dans le cerveau les zones-intégrative (Shien) - à partir du MEXT au Japon, et par une subvention en aide à partir crête de la Japan Science and Technology Agency.

Dissection de gyrus denté hippocampique <ol><li> Dans une souris profondément anesthésiés, soigneusement disséquer le cerveau hors du crâne et de le placer dans la glace froide tampon phosphate salin (PBS). </li><li> Dans une boîte de Petri contenant PBS glacé, coupé le cerveau le long de la fissure longitudinale du cerveau à l&#39;aide d&#39;un couteau chirurgical, et de couper les régions postérieures à lambda (mésencéphale, le rhombencéphale et le cervelet). </li><li> Placez le côté médial hémisphère cérébral et, en utilisant une pince, retirer délicatement le diencéphale (thalamus et l&#39;hypothalamus) sous un microscope de dissection. Cela permettra d&#39;exposer la face médiale de l&#39;hippocampe, permettant la visualisation du gyrus denté. Le gyrus denté se distingue de la corne d&#39;Ammon par les écarts entre eux. Blessure à l&#39;hippocampe ou dans les environs, il sera plus difficile d&#39;isoler le gyrus denté. </li><li> Insérez une forte aiguille à pointe (par exemple, de calibre 27-aiguille) dans chaque côté du gyrus denté (limites du gyrus denté et la corne d&#39;Ammon; figure 1), et faites glisser les aiguilles superficiellement le long de l&#39;axe septo-temporelle de l&#39;hippocampe à isoler le gyrus denté. </li><li> Ramassez le gyrus denté isolés à l&#39;aide d&#39;une aiguille ou une pince et le placer dans un tube d&#39;échantillon. Les échantillons ainsi obtenus denté tissus gyrus peut être utilisé immédiatement pour toute analyse ou stockés dans un congélateur pour une utilisation ultérieure. </li><li> Isoler le gyrus denté de l&#39;autre hémisphère cérébral en utilisant la même méthode. </li></ol> Quantitative PCR en temps réel Le gyrus denté a été isolé en utilisant la méthode ci-dessus et l&#39;hippocampe reste a été disséqué comme échantillon de corne d&#39;Ammon de souris de type sauvage. PCR en temps réel de la bêta-actine, TDO2, DSP, Mrg1b et Tyro3 ont été effectuées avec le gyrus denté et des échantillons de la corne d&#39;Ammon, comme décrit précédemment 1. Amorces 5&#39;-CTGGCGAGATCACGATGACG et 5&#39;-AAGCTACGCTGTTGTCTAACC ont été utilisés pour Mrg1b et GCCTCCAAATTGCCCGTCA et 5&#39;-CCAGCACTGGTACATGAGATCA pour Tyro3. L&#39;analyse des microréseaux Expériences biopuces ont été effectués avec des hommes souris de type sauvage et les souris hétérozygotes pour l&#39;alpha-isoforme de calcium / calmoduline-dépendante protéine kinase II (alpha-CaMKII + / - souris), comme décrit précédemment 1. En bref, l&#39;ARN isolé de l&#39;hippocampe tout ou gyrus denté de souris de type sauvage et mutant a été hybridée avec une matrice de souris Génome 430 2.0 (Affymetrix, Santa Clara, CA), et chaque GeneChip a été scannée par un scanner Affymetrix GeneChip 3000 (GCS3000) . L&#39;analyse a été réalisée avec GeneChip Microarray Analysis Suite version 5.0.

<ol>
	<li>Yamasaki, N., Maekawa, M., Kobayashi, K., Kajii, Y., Maeda, J., Soma, M., Takao, K., Tanda, K., Ohira, K., Toyama, K., Kanzaki, K., Fukunaga, K., Sudo, Y., Ichinose, H., Ikeda, M., Iwata, N., Ozaki, N., Suzuki, H., Higuchi, M., Suhara, T., Yuasa, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-CaMKII+deficiency+causes+immature+dentate+gyrus,+a+novel+candidate+endophenotype+of+psychiatric+disorders.">Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders.</a> Mol. Brain. 1, 6-6 (2008).</li><li>Insausti, A. M., Megias, M., Crespo, D., Cruz-Orive, L. M., Dierssen, M., Vallina, I. F., Insausti, R., Florez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+volume+and+neuronal+number+in+Ts65Dn+mice:+a+murine+model+of+Down+syndrome.">Hippocampal volume and neuronal number in Ts65Dn mice: a murine model of Down syndrome.</a> Neurosci. Lett. 253, 175-175 (1998).</li><li>Redwine, J. M., Kosofsky, B., Jacobs, R. E., Games, D., Reilly, J. F., Morrison, J. H., Young, W. G., Bloom, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dentate+gyrus+volume+is+reduced+before+onset+of+plaque+formation+in+PDAPP+mice:+a+magnetic+resonance+microscopy+and+stereologic+analysis.">Dentate gyrus volume is reduced before onset of plaque formation in PDAPP mice: a magnetic resonance microscopy and stereologic analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 1381-1381 (2003).</li><li>Lein, E. S., Zhao, X., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+molecular+atlas+of+the+hippocampus+using+DNA+microarrays+and+high-throughput+in+situ+hybridization.">Defining a molecular atlas of the hippocampus using DNA microarrays and high-throughput in situ hybridization.</a> J. Neurosci. 24, 3879-3879 (2004).</li><li>Matsuo, N., Yamasaki, N., Ohira, K., Takao, K., Toyama, K., Eguchi, M., Yamaguchi, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+activity+changes+underlying+the+working+memory+deficit+in+alpha-CaMKII+heterozygous+knockout+mice.">Neural activity changes underlying the working memory deficit in alpha-CaMKII heterozygous knockout mice.</a> Front. Behav. Neurosci. 3, 20-20 (2009).</li><li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li></ol>

Les expérimentations animales ont été effectuées en conformité avec l&#39;Institut National de Guide de santé pour les soins et l&#39;utilisation des animaux de laboratoire, et approuvé par le comité de soins des animaux et l&#39;utilisation à Fujita universitaire de santé.

Le gyrus denté occupe environ 25% à 30% du volume de la formation hippocampique 2,3. Il a une composition cellulaire unique et joue un rôle crucial dans les fonctions cérébrales différentes. Par conséquent, des techniques pour isoler le gyrus denté sont utiles pour analyser les événements qui se produisent précisément dans cette région. Ici, nous avons démontré une procédure efficace disséquer le gyrus denté de l&#39;hippocampe de souris adultes et confirmé la précision de la technique. Premièrement, l&#39;étude histologique a révélé que le gyrus denté a été séparé sans contamination par d&#39;autres régions (figure 1), indiquant que d&#39;un échantillon gyrus denté pur peut être préparé. Deuxièmement, nous avons confirmé que le tissu est disséqué gyrus denté en effectuant des PCR en temps réel du gyrus denté de gènes spécifiques, TDO2 et DSP, et Ammon Corne gènes enrichis, et Mrg1b Tyro3 4 (figure 2). Les expressions d&#39;ARNm de TDO2 (p = 0,000023; N = 4 et 4, respectivement) et DSP (p = 0,0000030; N = 4 et 4, respectivement) dans les échantillons gyrus denté ont été détectés à des niveaux bien évidemment plus élevé, alors que Mrg1b (p = 0.000080; N = 4 et 4, respectivement) et Tyro3 (p = 0,00017; N = 4 et 4, respectivement) ont été des niveaux inférieurs, à ceux dans les échantillons de la corne d&#39;Ammon. Les niveaux d&#39;expression de bêta-actine ne diffère pas dans ces échantillons (p = 0,11; N = 4 et 4, respectivement). Ainsi, nous pourrions vérifier si oui ou non le gyrus denté a été précisément disséqué par conducteur tel temps réel simple expériences de PCR. Troisièmement, afin d&#39;évaluer l&#39;utilité de cette méthode de dissection, nous avons comparé le niveau d&#39;expression d&#39;ARNm de l&#39;hippocampe entier avec celle du gyrus denté. Hippocampe entier et gyrus denté obtenu à partir du type sauvage (N = 9 et 4, respectivement) et l&#39;alpha-CaMKII + / - souris (= N 18 et 4, respectivement) ont été traitées pour l&#39;analyse des microréseaux, et pour tous les gènes marqués, le double changement a été calculée en divisant la valeur mutant par la valeur de type sauvage. Les résultats indiquent que les changements dans l&#39;expression des ARNm, en particulier du gyrus denté molécules spécifiques tels que DSP et TDO2, ont été détectés jusqu&#39;à une multiplication par 5 de la sensibilité dans les échantillons gyrus denté par rapport à l&#39;ensemble des échantillons hippocampique (tableau 1). Nous avons précédemment démontré que l&#39;alpha-CaMKII + / - souris présentent des comportements humains liés à des troubles psychiatriques tels que troubles de la mémoire de travail et un rythme exagérée infradien 1,5. Par ailleurs, les caractéristiques morphologiques et électrophysiologiques des neurones dans le gyrus denté des souris mutantes sont étonnamment semblables à celles des neurones immatures gyrus denté chez les rongeurs normaux, indiquant que les neurones dans ces souris mutantes ne parviennent pas à se développer jusqu&#39;à maturité 1. Les immatures l&#39;expression denté gyrus et régulé à la baisse de la DSP et TDO2 ARNm dans l&#39;alpha-CaMKII + / - souris sont compatibles avec la conclusion selon laquelle DSP et TDO2 peuvent être utilisés comme marqueurs de cellules granulaires matures dans le gyrus denté (Ohira et al, non publié. données). Pris ensemble, cette technique de dissection pratique et précis peut être utilisé de manière fiable pour des études axées sur le gyrus denté. Tissus gyrus denté obtenus en utilisant cette méthode est applicable à d&#39;autres types d&#39;analyses aussi bien, y compris les analyses de biologie cellulaire et en protéomique. <img src="/files/ftp_upload/1543/1543fig1.jpg" alt="Figure 1" /> Figure 1. Vérification du gyrus denté isolée par l&#39;étude histologique. Une coupe coronale du cerveau après avoir isolé gyrus denté a été traitée pour la coloration de Nissl (panneau de gauche), et un schéma adapté à partir du cerveau de souris représente la atlas6 approximativement le même niveau de la section représentée dans le panneau de gauche (panneau de droite). Les flèches indiquent les directions de l&#39;insertion de l&#39;aiguille à pointe. La barre d&#39;échelle, de 1 mm. <img src="/files/ftp_upload/1543/1543fig2.jpg" alt="Figure 2" /> Figure 2. Vérification du gyrus denté isolés par PCR en temps réel. Le gyrus denté et de la corne d&#39;Ammon s obtenus à partir de quatre souris de type sauvage ont été traitées pour PCR en temps réel de la bêta-actine, TDO2, DSP, Mrg1b et Tyro3. Les résultats sont présentés en moyenne ± SEM. Pour l&#39;analyse statistique, le test de Student t s a été employé, et les valeurs de p sont suivis: la bêta-actine, p = 0,11; TDO2, p = 0,000023 (** 1); Dsp, p = 0,0000030 (** 2); Mrg1b, p = 0.000080 (** 3); Tyro3, ​​p = 0,00017 (** 4). Tableau 1. . Analyse des microréseaux de l&#39;hippocampe et le gyrus denté ensemble des gènes exprimés différentiellement dans le gyrus dentelé et l&#39;hippocampe ensemble de l&#39;alpha-CaMKII + / - souris ont été déterminés en calculant le pli de changement de celle détectée dans les souris de type sauvage. Les données ont été analysées pour la signification statistique en utilisant le test t de Student s entre type sauvage et l&#39;alpha-CaMKII + / - souris. Parmi les gènes dont l&#39;expression exposées p &lt;0,05 dans lesle gyrus denté de l&#39;alpha-CaMKII + / - souris par rapport à celle des souris de type sauvage, les 50 premiers gènes sont répertoriés. Notez que le nombre d&#39;échantillons pour gyrus denté sont beaucoup moins que ceux pour l&#39;ensemble hippocampe. AffyID, Affymetrix sonde identifiant; CKII, l&#39;alpha-CaMKII + / - souris; WT, souris de type sauvage. <table border="1"><tbody><tr><td></td><td></td><td></td><td></td><td colspan="2"> Denté gyrus (p &lt;0,05) WT: n = 4, CKII + / -: n = 4 </td><td></td><td colspan="2"> Hippocampe entier WT: n = 9, CKII + / -: n = 18 </td></tr><tr><td> Titre Gene </td><td> Genebank </td><td> AffyID </td><td></td><td> Pliez le changement </td><td> valeur p </td><td></td><td> Pliez le changement </td><td> valeur p </td></tr><tr><td> desmoplakine </td><td> AV297961 </td><td> 1435494_s_at </td><td></td><td> 0,011018913 </td><td> 7.02694E-06 </td><td></td><td> 0,037021003 </td><td> 1.86126E-13 </td></tr><tr><td> desmoplakine </td><td> AV297961 </td><td> 1435493_at </td><td></td><td> 0,014369734 </td><td> 7.86747E-06 </td><td></td><td> 0.04232106 </td><td> 1.00579E-12 </td></tr><tr><td> tryptophane 2,3-dioxygénase </td><td> AI098840 </td><td> 1419093_at </td><td></td><td> 0,020986484 </td><td> 5.23546E-09 </td><td></td><td> 0,101037776 </td><td> 4.14823E-13 </td></tr><tr><td> nephronectin </td><td> AA223007 </td><td> 1452106_at </td><td></td><td> 0,075479901 </td><td> 1.05191E-08 </td><td></td><td> 0,234001154 </td><td> 1.66301E-15 </td></tr><tr><td> nephronectin </td><td> AA223007 </td><td> 1452107_s_at </td><td></td><td> 0,079457767 </td><td> 1.40433E-07 </td><td></td><td> 0,177974715 </td><td> 3.9758E-12 </td></tr><tr><td> thyrotropine libérant des récepteurs hormonaux </td><td> M59811 </td><td> 1449571_at </td><td></td><td> 0,103105815 </td><td> 0,003093796 </td><td></td><td> 0,801412732 </td><td> 0,283994361 </td></tr><tr><td> ryanodine récepteurs 1, le muscle squelettique </td><td> X83932 </td><td> 1427306_at </td><td></td><td> 0,104825517 </td><td> 3.38513E-07 </td><td></td><td> 0,650685017 </td><td> 0,000308462 </td></tr><tr><td> ignorant hélice boucle hélice 1 </td><td> NM_010916 </td><td> 1419533_at </td><td></td><td> 9.431896 </td><td> 6.7979E-06 </td><td></td><td> 4,078815314 </td><td> 5.27E-11 </td></tr><tr><td> COPINE membre de la famille IX </td><td> BB274531 </td><td> 1454653_at </td><td></td><td> 9.159157 </td><td> 7.99492E-06 </td><td></td><td> 1,797304153 </td><td> 0,000296375 </td></tr><tr><td> doublecortine-like kinase 3 </td><td> BB326709 </td><td> 1436532_at </td><td></td><td> 0,109336662 </td><td> 1.95278E-07 </td><td></td><td> 0.56697229 </td><td> 2.62633E-08 </td></tr><tr><td> calpaïne 3 </td><td> AF127766 </td><td> 1426043_a_at </td><td></td><td> 0,111269769 </td><td> 8.07053E-06 </td><td></td><td> 0,370956608 </td><td> 2.04421E-14 </td></tr><tr><td> Adulte striatum hommes corpus d&#39;ADNc, RIKEN pleine longueur enrichi la bibliothèque, clone: ​​C030023B07 produit: inclassable, pleine séquence de l&#39;insert </td><td> BB357628 </td><td> 1460043_at </td><td></td><td> 0,118712341 </td><td> 6.16926E-07 </td><td></td><td> 0,682339204 </td><td> 2.33001E-06 </td></tr><tr><td> collagène et la fixation du calcium FEM domaines 1 </td><td> AV264768 </td><td> 1437385_at </td><td></td><td> 0,124043978 </td><td> 3.65669E-05 </td><td></td><td> 0,488394112 </td><td> 4.05538E-06 </td></tr><tr><td> bêta-amyloïde (A4) précurseur de liaison aux protéines, la famille A, membre 2 de liaison aux protéines </td><td> AK013520 </td><td> 1431946_a_at </td><td></td><td> 7.7986307 </td><td> 1.2098E-06 </td><td></td><td> 2,099164713 </td><td> 1.67047E-06 </td></tr><tr><td> calbindine 28K </td><td> BB177770 </td><td> 1456934_at </td><td></td><td> 0,130255444 </td><td> 3.32186E-06 </td><td></td><td> 0,572605751 </td><td> 1.99157E-10 </td></tr><tr><td> Le locus transcrit </td><td> AV328597 </td><td> 1443322_at </td><td></td><td> 0,133290835 </td><td> 5.43583E-06 </td><td></td><td> 0,562767164 </td><td> 7.56544E-06 </td></tr><tr><td> neuropeptide Y Y2 récepteurs </td><td> NM_008731 </td><td> 1417489_at </td><td></td><td> 0,135319609 </td><td> 0,000113407 </td><td></td><td> 0,781498474 </td><td> 0.00394504 </td></tr><tr><td> protéine Ras responsive element binding 1 </td><td> BE197381 </td><td> 1428657_at </td><td></td><td> 0,138235114 </td><td> 7.93691E-07 </td><td></td><td> 0,651220705 </td><td> 2.94209E-05 </td></tr><tr><td> la ligne neurotrophique dérivé des cellules gliales famille des récepteurs des facteurs alpha 2 </td><td> BB284482 </td><td> 1433716_x_at </td><td></td><td> 0,139062563 </td><td> 2.35371E-06 </td><td></td><td> 0,669544709 </td><td> 0,000214146 </td></tr><tr><td> préproenképhaline 1 </td><td> M13227 </td><td> 1427038_at </td><td></td><td> 6.9850435 </td><td> 2.39074E-08 </td><td></td><td> 1,766018828 </td><td> 0,000250501 </td></tr><tr><td> RIKEN gène ADNc 1810010H24 </td><td> BI729991 </td><td> 1428809_at </td><td></td><td> 6.8658915 </td><td> 1.88516E-05 </td><td></td><td> 2.77573142 </td><td> 6.81865E-09 </td></tr><tr><td> ryanodine récepteurs 1, le muscle squelettique </td><td> BG793713 </td><td> 1457347_at </td><td></td><td> 0,151364292 </td><td> 3.35612E-05 </td><td></td><td> 0,503144617 </td><td> 4.32907E-05 </td></tr><tr><td> protocadhérine 21 </td><td> NM_130878 </td><td> 1418304_at </td><td></td><td> 0,152671849 </td><td> 8.57783E-06 </td><td></td><td> 0,670714726 </td><td> 1.56309E-05 </td></tr><tr><td> homologue cornichon 3 (Drosophila) </td><td> NM_028408 </td><td> 1419517_at </td><td></td><td> 0,153724144 </td><td> 8.90755E-06 </td><td></td><td> 0.95780695 </td><td> 0,661055608 </td></tr><tr><td> harakiri, BCL2 protéines en interaction (ne contient que BH3 domaine) </td><td> BQ175572 </td><td> 1439854_at </td><td></td><td> 0,154284407 </td><td> 2.0118E-05 </td><td></td><td> 0.56516812 </td><td> 4.86925E-09 </td></tr><tr><td> glucides (N-acétyl 4-0) sulfotransférase 9 </td><td> AK017407 </td><td> 1431897_at </td><td></td><td> 0,155238951 </td><td> 5.37423E-06 </td><td></td><td> 1.14910007 </td><td> 0,215637733 </td></tr><tr><td> calpaïne 3 </td><td> AI323605 </td><td> 1433681_x_at </td><td></td><td> 0,160871988 </td><td> 1.07655E-05 </td><td></td><td> 0,477164757 </td><td> 1.33753E-11 </td></tr><tr><td> doigt de zinc, de domaine contenant 5 CCHC </td><td> BQ126004 </td><td> 1437355_at </td><td></td><td> 0,161812078 </td><td> 3.08262E-06 </td><td></td><td> 0,421252632 </td><td> 0.01152969 </td></tr><tr><td> loricrine </td><td> NM_008508 </td><td> 1448745_s_at </td><td></td><td> 0,165129967 </td><td> 1.86362E-05 </td><td></td><td> 0,639733409 </td><td> 0,000729772 </td></tr><tr><td> spondine 1, (f-spondine) des protéines de la matrice extracellulaire </td><td> BC020531 </td><td> 1451342_at </td><td></td><td> 0,168035879 </td><td> 6.67867E-07 </td><td></td><td> 0,821042412 </td><td> 0,023650765 </td></tr><tr><td> RIKEN ADNc du gène A930035E12 </td><td> AV348640 </td><td> 1429906_at </td><td></td><td> 5.9086795 </td><td> 1.747E-07 </td><td></td><td> 1,470383201 </td><td> 0,104085454 </td></tr></tbody><tbody><tr><td> BB247294 RIKEN pleine longueur enrichi, 7 jours cervelet nouveau-né Mus musculus clone d&#39;ADNc A730018G18 3 &#39;, la séquence d&#39;ARNm. </td><td> BB247294 </td><td> 1447907_x_at </td><td></td><td> 5.9047494 </td><td> 1.04931E-05 </td><td></td><td> 1,968147585 </td><td> 0.00010636 </td></tr><tr><td> Domaine FERM contenant 3 </td><td> BB099015 </td><td> 1437075_at </td><td></td><td> 5.860216 </td><td> 0,000345581 </td><td></td><td> 2,780297178 </td><td> 1.83072E-06 </td></tr><tr><td&gt; Neuronale pentraxine 2 / / / protéine hypothétique LOC100044234 </td><td> NM_016789 </td><td> 1420720_at </td><td></td><td> 5.7568517 </td><td> 1.34227E-06 </td><td></td><td> 2,652516957 </td><td> 0,000206279 </td></tr><tr><td> Séquences transcrites </td><td> BG076361 </td><td> 1460101_at </td><td></td><td> 5.657735 </td><td> 2.5015E-06 </td><td></td><td> 1,296248831 </td><td> 0.22870031 </td></tr><tr><td> spondine 1, (f-spondine) des protéines de la matrice extracellulaire </td><td> BC020531 </td><td> 1424415_s_at </td><td></td><td> 0.17783576 </td><td> 1.01658E-06 </td><td></td><td> 0,836181248 </td><td> 0,001380141 </td></tr><tr><td> calbindine 28K </td><td> BB246032 </td><td> 1448738_at </td><td></td><td> 0,180317904 </td><td> 1.35961E-05 </td><td></td><td> 0,647334052 </td><td> 4.12268E-09 </td></tr><tr><td> MARCKS-like 1 </td><td> AV110584 </td><td> 1437226_x_at </td><td></td><td> 0,186235935 </td><td> 1.47067E-06 </td><td></td><td> 0,499291387 </td><td> 2.34984E-08 </td></tr><tr><td> matrilin 2 </td><td> BB338441 </td><td> 1455978_a_at </td><td></td><td> 0,187783528 </td><td> 6.19122E-05 </td><td></td><td> 0.8967688 </td><td> 0,282337853 </td></tr><tr><td> matrilin 2 </td><td> BC005429 </td><td> 1419442_at </td><td></td><td> 0,188195795 </td><td> 0,000105295 </td><td></td><td> 0,915528892 </td><td> 0.35282097 </td></tr><tr><td> spondine 1, (f-spondine) des protéines de la matrice extracellulaire </td><td> BQ175871 </td><td> 1442613_at </td><td></td><td> 0,189956563 </td><td> 9.41195E-06 </td><td></td><td> 0,861033222 </td><td> 0.1394266 </td></tr><tr><td> arrestine 3, la rétine </td><td> NM_133205 </td><td> 1450329_a_at </td><td></td><td> 5.2130346 </td><td> 2.90599E-05 </td><td></td><td> 3,944218329 </td><td> 1.07437E-07 </td></tr><tr><td> RIKEN ADNc du gène A330050F15 </td><td> AV325555 </td><td> 1457558_at </td><td></td><td> 0.19186781 </td><td> 0,000119342 </td><td></td><td> 0,660282035 </td><td> 2.47342E-05 </td></tr><tr><td> contactin 3 </td><td> BB559510 </td><td> 1438628_x_at </td><td></td><td> 0,194404608 </td><td> 4.08641E-07 </td><td></td><td> 0,918742591 </td><td> 0,022545297 </td></tr><tr><td> calbindine 28K </td><td> BB246032 </td><td> 1417504_at </td><td></td><td> 0,196381321 </td><td> 2.24182E-05 </td><td></td><td> 0,619305124 </td><td> 3.6222E-06 </td></tr><tr><td> libération de la gastrine peptide </td><td> BC024515 </td><td> 1424525_at </td><td></td><td> 4.9436426 </td><td> 3.00588E-05 </td><td></td><td> 2,752845903 </td><td> 5.72954E-07 </td></tr><tr><td> Sortiline liés de domaine contenant des récepteurs VPS10 3 </td><td> AK018111 </td><td> 1425111_at </td><td></td><td> 4.885766 </td><td> 1.03645E-05 </td><td></td><td> 1.29051599 </td><td> 0,029733649 </td></tr><tr><td> récepteur de la dopamine D1A </td><td> BE957273 </td><td> 1455629_at </td><td></td><td> 4.869493 </td><td> 3.77525E-05 </td><td></td><td> 1,815881979 </td><td> 0,000516498 </td></tr><tr><td> proprotéine convertase subtilisine / Kexin type 5 </td><td> BB241731 </td><td> 1437339_s_at </td><td></td><td> 0,210528027 </td><td> 7.83039E-05 </td><td></td><td> 0,574126078 </td><td> 9.15496E-05 </td></tr></tbody><tbody><tr><td> récepteurs d&#39;interleukine 1, de type I </td><td> NM_008362 </td><td> 1448950_at </td><td></td><td> 0,210572243 </td><td> 9.64524E-06 </td><td></td><td> 0,241135352 </td><td> 2.79816E-08 </td></tr></tbody></table>

dissection of hippocampal dentate gyrus from adult mouse

L&#39;hippocampe est l&#39;une des zones les plus étudiées dans le cerveau en raison de son rôle fonctionnel important dans le traitement de la mémoire et l&#39;apprentissage, sa remarquable plasticité des cellules neuronales, et son implication dans l&#39;épilepsie, les maladies neurodégénératives et les troubles psychiatriques. L&#39;hippocampe est composé de régions distinctes; le gyrus denté, qui comprend principalement les neurones granulaires, et la corne d&#39;Ammon, qui comprend principalement des neurones pyramidaux, et les deux régions sont reliées par deux circuits anatomiques et fonctionnelles. Beaucoup de différents ARNm et des protéines sont exprimées sélectivement dans le gyrus denté, et le gyrus denté est un site de la neurogenèse adulte, c&#39;est de nouveaux neurones sont continuellement générés dans le gyrus denté pour adultes. Afin d&#39;étudier l&#39;expression des ARNm et des protéines spécifiques pour le gyrus denté, microdissection laser est souvent utilisé. Cette méthode a toutefois quelques limitations, comme la nécessité d&#39;appareils spéciaux et des procédures de manipulation compliquée. Dans ce protocole d&#39;enregistrements vidéo, nous démontrons une technique de dissection pour enlever le gyrus denté de souris adulte sous un stéréomicroscope. Échantillons gyrus denté préparés en utilisant cette technique sont adaptés à toute épreuve, y compris la transcriptomique, la protéomique, et les analyses de biologie cellulaire. Nous avons confirmé que le tissu est disséqué gyrus denté en effectuant des PCR en temps réel du gyrus denté de gènes spécifiques, le tryptophane 2,3-dioxygénase (TDO2) et desmoplakine (DSP), et d&#39;Ammon Corne gènes enrichis, 1b du gène Meis-connexes (Mrg1b ) et TYRO3 protéine tyrosine kinase 3 (Tyro3). Les expressions des ARNm TDO2 et DSP dans les échantillons gyrus denté ont été détectés à des niveaux plus élevés de toute évidence, alors que Mrg1b et Tyro3 ont des niveaux plus bas que ceux dans les échantillons de la corne d&#39;Ammon. Afin de démontrer l&#39;avantage de cette méthode, nous avons effectué une analyse des microréseaux d&#39;ADN en utilisant des échantillons de l&#39;hippocampe et le gyrus denté entière. L&#39;expression de l&#39;ARNm du TDO2 et DSP, qui sont exprimés de manière sélective dans le gyrus denté, dans l&#39;hippocampe ensemble de l&#39;alpha-CaMKII + / - souris, exposées 0,037 et 0,10 fois les changements par rapport à celle des souris de type sauvage, respectivement. Dans le gyrus denté isolée, cependant, ces expressions exposées 0,011 et 0,021 fois changements par rapport à celle des souris de type sauvage, ce qui démontre que les changements d&#39;expression génique dans le gyrus denté peuvent être détectés avec une plus grande sensibilité. Pris ensemble, cette technique de dissection pratique et précis peut être utilisé de manière fiable pour des études axées sur le gyrus denté.

Watch this Scientific Journal Video about Dissection of Hippocampal Dentate Gyrus from Adult Mouse at JoVE.com

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

Une technique de dissection de l&#39;enlèvement du gyrus denté de souris adulte sous un stéréomicroscope a été démontrée dans ce protocole d&#39;enregistrements vidéo.

Dissection de l&#39;hippocampe gyrus denté de souris adulte

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

dissection-of-hippocampal-dentate-gyrus-from-adult-mouse

Research

JoVE Journal

Biology

82.2K vues.  Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST). Une technique de dissection de l'enlèvement du gyrus denté de souris adulte sous un stéréomicroscope a été démontrée dans ce protocole d'enregistrements vidéo.

Vidéo: Dissection of Hippocampal Dentate Gyrus from Adult Mouse

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Dissection de 6,5 embryons de souris DPC

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Recherche

Biologie

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Dissection d&#39;organes sur le poisson zèbre pour adultes

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Dissection de Oenocytes d&#39;adultes Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Dissection du rein Zebrafish adultes

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Dissection du cœur chez les larves, le poisson zèbre juvéniles et adultes, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

Génération de souris dérivées de cellules souches pluripotentes induites

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

Isolation et culture des kératinocytes de la souris primaire de la peau de souris néonatale et adulte

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Isolement, caractérisation et différenciation des cellules souches cardiaques du coeur de souris adulte

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...

Isolation and Culture of Primary Oral Keratinocytes from the Adult Mouse Palate

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have attracted attention because of their unique function and characteristics. They maintain the homeostasis of the oral epithelium and serve as resources for applications in regenerative therapies. However, in vitro studies that use oral primary keratinocytes from adult mice have been limited due to the lack of an efficient and well-established culture protocol. Here, oral primary keratinocytes were isolated from the palate tissues of adult mice and cultured in a commercial low-calcium medium supplemented with a chelexed-serum. Under these conditions, keratinocytes were maintained in a proliferative or stem cell-like state, and their differentiation was inhibited even after increased passages. Marker expression analysis showed that the cultured oral keratinocytes expressed the basal cell markers p63, K14, and &#945;6-integrin and were negative for the differentiation marker K13 and the fibroblast marker PDGFR&#945;. This method produced viable and culturable cells suitable for downstream applications in the study of oral epithelial stem cell functions in vitro.

The present protocol describes the isolation and culture of oral keratinocytes derived from the adult mouse palate. An evaluation method using immunostaining is also reported.

Isolement et culture de kératinocytes oraux primaires du palais de souris adulte

For years, most studies involving keratinocytes have been conducted using human and mouse skin epidermal keratinocytes. Recently, oral keratinocytes have ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.