Biz filme destek için diseksiyon tekniği ve Fujita Sağlık Üniversitesi Bayan Aki Miyakawa onu talimat Kyoto Üniversitesi Dr. Yoko Nabeshima teşekkür ederim. Japonya&#39;da MEXT gelen, ve - Bu çalışma, Ulusal Biyomedikal Yenilik, Öncelik Alanları-Bütünleştirici Beyin Araştırmaları (SHIEN) Bilimsel Araştırma için Hibe Yardım Enstitüsü Sağlık Bilimleri Temel Araştırmalar Teşvik Programı tarafından desteklenmiştir. Hibe-Japonya Bilim ve Teknoloji Kurumu CREST-Aid.

Hipokampal dentat girus diseksiyonu <ol><li> Derin bir anestezi fare, kafatası dikkatlice beyni incelemek ve buz fosfat tamponlu salin (PBS) içine yerleştirin. </li><li> Buz gibi soğuk PBS içeren bir Petri kabındaki beyin, beyin cerrahi bir bıçak kullanarak uzunlamasına fissür boyunca kesme ve lambda posterior bölgeler (orta beyin, arka beyin ve beyincik) kesti. </li><li> Serebral hemisferin medial yüzü yukarı bakacak ve forseps kullanarak, dikkatli bir diseksiyon mikroskobu altında diensefalon (talamus ve hipotalamus) çıkarın. Bu dentat girus görünüm için sağlayarak, hipokampus, medial tarafında gösterecektir. Dentat girus aralarındaki boşluklar Ammon boynuzu ayırt. Hipokampus yaralanma veya çevresini, dentat girus izole etmek için daha zor hale getirecektir. </li><li> Dentat girus dentat girus ve Ammon Kullanıcı boynuzu sınırları; Şekil 1&#39;de her tarafında içine bir keskin iğne-ucu (örneğin, 27-ölçer iğne) takın, ve izole etmek için hipokampüsün septo-zamansal ekseni boyunca yüzeysel iğneler slayt dentat girus. </li><li> Bir iğne veya forseps kullanarak izole dentat girus Pick up ve örnek bir tüp içine koyun. Böylece elde dentat girus doku örneği hemen her tayini için kullanılan ya da daha sonra kullanmak üzere derin dondurucuda saklanabilir. </li><li> Aynı yöntemi kullanarak diğer serebral hemisferin dentat girus ayırın. </li></ol> Kantitatif Gerçek-zamanlı PCR Dentat girus, yukarıda belirtilen yöntem ve kalan hipokampüs Ammon boynuzu, wild-tip farelerden alınan örnek olarak diseke edildi kullanılarak izole edilmiştir. Dentat girus ve daha önce 1 açıklandığı gibi Ammon boynuz örnekleri beta-aktin, TDO2, DSP, Mrg1b ve Tyro3 Real-time PCR ile yapıldı. Astarlar 5&#39;-CTGGCGAGATCACGATGACG ve 5&#39;-AAGCTACGCTGTTGTCTAACC Tyro3 Mrg1b ve GCCTCCAAATTGCCCGTCA ve 5&#39;-CCAGCACTGGTACATGAGATCA için kullanılmıştır. Mikroarray analizi (- Fareler alpha-CaMKII + /) daha önce 1 açıklanan Mikroarray deneyler erkek wild-tip farelerden ve kalsiyum / kalmodulin-bağımlı protein kinaz II alfa-izoformu için heterozigot farelerde ile yapıldı. Kısaca, yabani tip ve mutant farelerin tüm hipokampus ve dentat girus izole RNA Fare Genom 430 2.0 Array (Affymetrix, Santa Clara, CA) ile melezleşmiştir, ve her GeneChip Affymetrix GeneChip Scanner 3000 (GCS3000) tarafından taranan . GeneChip analiz Mikroarray Analizi Suite sürümü 5.0 ile yapıldı.

<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>

Hayvan deneyleri Ulusal Enstitüsü Laboratuvar Hayvanları Bakım ve Kullanım Sağlık Rehberi uyarınca yapılır ve Fujita Sağlık Üniversitesi&#39;nde hayvan bakımı ve kullanımı komitesi tarafından kabul edildi.

Dentat girus hipokampal formasyon 2,3 hacmi yaklaşık% 25 -% 30 kaplar . Bu eşsiz bir hücre kompozisyon ve çeşitli beyin fonksiyonlarında önemli roller oynamaktadır. Bu nedenle, dentat girus izole teknikleri özellikle bu bölgede meydana gelen olayları analiz etmek için yararlıdır. Burada, yetişkin fare hipokampus dentat girus verimli bir şekilde incelemek için bir prosedür gösterilmiş ve tekniği hassas doğruladı. İlk olarak, histolojik çalışma dentat girus, diğer bölgelere göre kontaminasyon (Şekil 1) olmadan, saf bir dentat girus örnek hazırlanmış olabilir belirten ayrılmış olduğunu ortaya çıkardı. İkincisi, disseke doku dentat girus özel genler, TDO2 ve DSP ve Ammon boynuzu zenginleştirilmiş genler, Mrg1b ve Tyro3 4 (Şekil 2) gerçek-zamanlı PCR yaparak dentat girus olduğunu doğruladı. TDO2, mRNA ifadeleri (p = 0,000023; n üyesinin = 4 ve 4, sırasıyla) ve DSP (p = 0,0000030; n üyesinin = 4 ve 4,) dentat girus örnekleri, tabii ki daha yüksek seviyelerde tespit edildi ise Mrg1b (p = 0.000080; n = 4 ve 4, sırasıyla) ve Tyro3 (p = 0,00017; n = 4 ve 4), Ammon boynuzu örnekleri daha düşük seviyelerde. Beta-aktin ekspresyon düzeyleri (n = 4 ve 4, p = 0.11), bu örneklerin farklı değildi . Böylece, dentat girus gibi basit bir gerçek-zamanlı PCR deneyler yaparak doğru disseke olup olmadığı kontrol edebilir. Bu diseksiyon yöntemi yararını değerlendirmek için, Üçüncü, dentat girus ile tüm hipokampus mRNA düzeyi karşılaştırıldı. Farelerde (sırasıyla n üyesinin = 18 ve 4,) mikroarray analizi için işlenmiş edildi ve tüm genlerin gol için, kat-- wild-tip (N = 9 ve 4, sırasıyla) ve alfa-CaMKII + / elde Tüm hipokampus ve dentat girus değişiklik yabani tip değer mutant değeri bölünmesi ile hesaplanır. Sonuçları, özellikle DSP ve TDO2 olarak dentat girus özel moleküllerin mRNA değişiklikler, dentat girus örnekleri duyarlılığı tüm hipokampal örnekleri (Tablo 1) göre 5 kat artış tespit edildi. Daha önce fareler sergi davranışları gibi çalışan bellek kusuru ve abartılı bir infradian ritim 1,5 gibi insan psikiyatrik bozukluklar ile ilgili alpha-CaMKII + / göstermiştir. Ayrıca, mutant farelerde dentat girus nöronlar morfolojik ve elektrofizyolojik özellikleri bu mutant farelerde nöron vadesi 1 geliştirmek için başarısız olduğunu belirten, bu normal kemirgenlerde olgunlaşmamış dentat girus nöronların şaşırtıcı bir benzerlik taşımaktadır. Olgunlaşmamış dentat girus ve down regüle DSP ifade ve alpha-CaMKII TDO2 mRNA + / - fareler DSP ve TDO2 dentat girus (Ohira et al olgun granül hücreleri belirteçleri olarak kullanılan olarak yapabilirsiniz bu bulgu ile tutarlı olan, yayınlanmamış. veri). Birlikte ele alındığında, bu uygun ve doğru diseksiyon tekniği güvenilir dentat girus odaklanmış çalışmaları için kullanılacak. Bu yöntemi kullanarak elde dentat girus doku proteomik ve hücre biyolojisi analizleri de dahil olmak üzere iyi analiz diğer türleri için geçerli. <img src="/files/ftp_upload/1543/1543fig1.jpg" alt="Şekil 1" /> Şekil 1 histolojik çalışmada izole dentat girus doğrulanması. Dentat girus izole sonra beynin koronal bölümünde Nissl boyama (sol panel), işlenmiş ve fare beyin atlas6 uyarlanmış bir şema ile sol paneli (sağ panel) gösterilen bölümün yaklaşık olarak aynı seviyede temsil eder. Oklar iğne ucu ekleme yöne işaret etmektedir. Ölçeği bar, 1 mm. <img src="/files/ftp_upload/1543/1543fig2.jpg" alt="Şekil 2" /> Şekil 2, gerçek zamanlı PCR ile izole dentat girus doğrulanması . Dentat girus ve Ammon s boynuz dört wild-tip farelerden elde edilen gerçek zamanlı beta-aktin PCR TDO2, DSP, Mrg1b ve Tyro3 için işlendi. Sonuçlar ortalama ± SEM olarak verilmiştir. İstatistiksel analiz için Student t testi kullanılmıştır ve p değerleri takip edilir: beta-aktin, p = 0.11; TDO2, p = 0,000023 (** 1); DSP, p = 0,0000030 (** 2); Mrg1b, p = 0.000080 (** 3) ve Tyro3, ​​p = 0,00017 (** 4). Tablo 1. Tüm hipokampus ve dentat girus Mikroarray analizi Genler farklı alfa-CaMKII dentat girus ve bütün hipokampus ifade + / - farelerde, wild-tip farelerden tespit kat değişim hesaplanarak belirlendi . Fareler - Veri yabani tip ve alpha-CaMKII + / arasında Student t testi kullanılarak istatistiksel anlamlılık için analiz edildi. Genler arasında p &lt;0.05 olan ifade sergiledialpha-CaMKII + / dentat girus - wild-tip farelerden göre fareler, ilk 50 gen listelenmiştir. Dentat girus için numune sayısının tüm hipokampus oranla çok daha az olduğunu unutmayın. AffyID, Affymetrix prob tanımlayıcısı; CKII, alpha-CaMKII + / - farelerde; WT, wild-tip farelerden. <table border="1"><tbody><tr><td></td><td></td><td></td><td></td><td colspan="2"> Dentat girus (p &lt;0.05) WT: n = 4, CKII + / -: n = 4 </td><td></td><td colspan="2"> Tüm hipokampus WT: n = 9, CKII + / -: n = 18 </td></tr><tr><td> Gen Başlığı </td><td> Genebank </td><td> AffyID </td><td></td><td> Katlama değişiklik </td><td> p değeri </td><td></td><td> Katlama değişiklik </td><td> p değeri </td></tr><tr><td> desmoplakin </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> desmoplakin </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> triptofan 2,3-dioxygenase </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> tirotropin hormonu salgılatıcı reseptör </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 reseptör 1, iskelet kası </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> bilgisiz sarmal döngü sarmal 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 aile üyesi 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> doublecortin-3 kinaz </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> calpain 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> Yetişkin erkek korpus striatum cDNA, RIKEN tam uzunlukta zenginleştirilmiş kütüphane, klon: C030023B07 ürün: unclassifiable, tam uç sırası </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> kolajen ve kalsiyum bağlayıcı EGF etki 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> amiloid beta (A4) prekürsör protein bağlama, aile, üye 2 bağlayıcı protein </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> Calbindin-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> Transkripsiyonu lokus </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> nöropeptid Y reseptör Y2 </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> ras duyarlı element bağlayıcı protein 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> glial hücre hattı nörotrofik faktör aile reseptörü elde edilen alfa 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> preproenkephalin 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 cDNA 1810010H24 gen </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 reseptör 1, iskelet kası </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> protocadherin 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> cornichon homolog 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 etkileşen protein (sadece Bh3 etki alanı içerir) </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> karbonhidrat (9 N-acetylgalactosamine 4-0) sulfotransferase </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> calpain 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> çinko parmak, CCHC etki alanı içeren 5 </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> loricrin </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> spondin 1 (f-spondin), ekstrasellüler matriks proteini </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 cDNA A930035E12 gen </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 tam uzunlukta zenginleştirilmiş 7 gün, yenidoğan beyincik Mus musculus cDNA klonu A730018G18 3 &#39;mRNA sırası. </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> Ferm etki alanı içeren 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; Nöronal pentraxin 2 / / / varsayımsal bir protein 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> Transkripsiyonu sekansları </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> spondin 1 (f-spondin), ekstrasellüler matriks proteini </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> Calbindin-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> 1 Marcks gibi </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> spondin 1 (f-spondin), ekstrasellüler matriks proteini </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> arrestin 3, retina </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 cDNA A330050F15 gen </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> Calbindin-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> gastrin peptid serbest </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> sortilin ilgili VPS10 etki alanı reseptör 3 içeren </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> dopamin reseptör 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> proprotein konvertaz subtilisin / kexin tip 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> interlökin 1 reseptör, tip 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

Hipokampus, beynin bellek işleme ve öğrenme önemli fonksiyonel rolü, olağanüstü nöronal hücre plastisite ve katılımının epilepsi, nörodejeneratif hastalıklar ve psikiyatrik bozukluklar nedeniyle en çok araştırılan alanlardan biridir. Hipokampus, farklı bölgelerde oluşur; özellikle granül nöronlar, özellikle piramidal nöronlar oluşur Ammon boynuzu, ve iki bölge oluşur dentat girus, hem anatomik ve fonksiyonel devreleri ile bağlıdır. Birçok farklı mRNA&#39;ların ve proteinler seçici dentat girus ifade ve dentat girus yetişkin nöron bir site olduğunu, sürekli yeni nöron yetişkin dentat girus oluşturulur. Dentat girus spesifik mRNA ve protein ifade araştırmak için, lazer yakalama mikrodiseksiyon sıklıkla kullanılmaktadır. Bu yöntem, özel aparatlar ve karmaşık işlemleri için ihtiyacınız olan, ancak bazı sınırlamalar vardır. Bu videoya kaydedilmiş protokol, stereomicroscope altında erişkin fareden dentat girus kaldırmak için bir diseksiyon tekniği göstermektedir. Bu tekniği kullanılarak hazırlanan dentat girus örnekleri, transcriptomic, proteomik ve hücre biyolojisi analizleri de dahil olmak üzere herhangi bir tahlil için uygundur. Disseke doku gerçek zamanlı dentat girus spesifik genlerin PCR, triptofan 2,3-dioxygenase (TDO2) ve desmoplakin (DSP) ve Ammon boynuzu zenginleştirilmiş genler, Meis ilgili gen 1b (Mrg1b yaparak dentat girus olduğunu doğruladı ) ve TYRO3 protein tirozin kinaz 3 (Tyro3). Mrg1b ve Tyro3 Ammon boynuzu örnekleri dışında, daha düşük düzeylerde iken, dentat girus örneklerinde TDO2 ve DSP mRNA ifadeleri, tabii ki daha yüksek seviyelerde tespit edildi. Bu yöntemin avantajı göstermek için, biz bütün hipokampus ve dentat girus örnekleri kullanarak DNA mikroarray analizi yapıldı. TDO2 ve DSP mRNA alpha-CaMKII + / tüm hipokampus, dentat girus seçici olarak ifade edilir, fareler, sırasıyla 0.037 ve wild-tip fareler ile karşılaştırıldığında 0.10 kat değişiklikler sergiledi. Izole dentat girus, ancak, bu ifadeleri daha büyük bir hassasiyet ile tespit edilebilir dentat girus bu gen ekspresyon değişiklikleri gösteren, 0.011 ve yabani tip farelerin göre 0.021 kat değişiklikler sergilenmektedir. Birlikte ele alındığında, bu uygun ve doğru diseksiyon tekniği güvenilir dentat girus odaklanmış çalışmaları için kullanılacak.

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

Bu videoya kaydedilmiş protokol stereomicroscope altında erişkin fareden dentat girus kaldırılması için diseksiyon tekniği gösterildi.

Yetişkin Mouse Hipokampal dentat girus diseksiyonu

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 Görüntüleme.  Japan Science and Technology Agency, Core Research for Evolutionary Science and Technology (CREST). Bu videoya kaydedilmiş protokol stereomicroscope altında erişkin fareden dentat girus kaldırılması için diseksiyon tekniği gösterildi.

Video: 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]).

6.5 DPC Fare Embriyolar diseksiyonu

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

Biyoloji

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.

Yetişkin Zebra balığı Organları diseksiyonu

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.

Yetişkin gelen Oenocytes diseksiyonu 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.

Yetişkin Zebra balığı Böbrek diseksiyonu

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.

Larvaların Kalp Diseksiyon, Çocuk ve Yetişkin Zebra balığı, 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.

İsteyerek Pluripotent Kök Hücrelerinin Elde Edilen Fare Üretimi

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.

Yenidoğan ve Erişkin Fare Ciltlerindeki Primer Fare Keratinositlerin İzolasyonu ve Kültürü

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.

Yalıtım, karakterizasyonu ve yetişkin fare kalp kalp kök hücre farklılaşma

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.

Yetişkin Fare Damak Tadından Birincil Oral Keratinositlerin İzolasyonu ve Kültürü

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.