Work in the authors' laboratories was supported by the UK Biotechnology and Biological Sciences Research Council (BB/J006602/1 and BB/L003236/1), the Wellcome Trust (WT100269MA) and the European Research Council (ERC, "Ideas" Programme 293544). SJ was supported by an EMBO long-term fellowship. The authors thank Stephen Grant for his technical assistance.

Etik Beyanı: Fare hayvancılık ve taşıma Hayvanlar (Bilimsel Prosedürler), Birleşik Krallık ve Değişiklik Yönetmeliği 2012 Yasası 1986 ile uyumlu, UK Home Office düzenlemeleri ve UCL etik komite kurallarına uygundur. NOT: Tüm çözümler dietil pyrocarbonate (DEPC) ile yapılmalıdır - herhangi bir kalıntı RNAaz ari yok etmek için arıtılmış su. tüm DEPC globülleri sonra kayboldu DEPC aşağılamak için otoklav kadar DEPC tedavi için, DEPC (litre başına 1 ml) ekleyin, şiddetle çalkalanır. 1. RNA Probe Sentezi <ol><li> DİKKAT: formamid tutarken, Kişisel Koruyucu Ekipman (KKE) aşınma ve güvenlik kabini kullanın. Hibridizasyon tamponu hazırlayın: DEPC ile muamele edilmiş% 50 (h / h) deiyonize formamid, mM Tris-HCI, pH 7.5, 200 mM NaCI, 5 mM EDTA, 10 ile, iyonu giderilmiş suyun 5 mM NaH 2 PO 4, 5 mM Na-2 HPO 4, 0.01 mg / ml maya tRNA, 1 x Denhardt solutiilgili ve% 10 / h dekstran sülfat ağ. </li><li> RNA polimeraz promotörüne sahip bir plazmid olarak, ilgi konusu genin cDNA dizisini ihtiva eden bir DNA klonu seçin. Bu örnek için, Aspa için T7 ve SP6 RNA polimeraz promoterleri (Ek Şekil 1) sahip bir pCMV-SPORT6 klon, IRAVp968C0654D (Genbank accessionBC024934) kullanın. </li><li> şablon olarak doğrusallaştırılmış plazma DNA hazırlanır. <ol><li> DNA klonunun büyütün T7 ve SP6 evrensel primerler ile üreticinin protokolüne ve sırasına göre, küçük-ölçekli bir plazmid arındırma kiti ile plazmid ekstrakte edin. </li><li> cDNA&#39;nın yönlü sarmal kolunun 5 &#39;ucunda kesen bir sınırlama enzimi ile 1.020 | ig plazmid DNA Digest. Bu örnek için, pCMV-SPORT6- Aspa plazmid sindirimi 100 birim SalI kullanın. 37 o C&#39;de 1.5 saat süreyle inkübe edin </li><li> plazmid tamamen doğrusallaştırılmış olup olmadığını kontrol etmek bir agaroz jel üzerinde küçük bir kısım çalıştırın. </li></ol></li><li> arındırmakfenol-kloroform kullanılarak doğrusallaştırılmış plazmit kullanmak avantajlıdır. <ol><li> 3 M sodyum asetatın 1/10 hacmi, 10 mM Tris HCI (pH 8.0), bir hacim ekleyin - dengelenmiş fenol ve kloroform / izoamil alkol (IAA), bir hacim (24: 1). </li><li> (- 25 ° C, RT 20) ve özü, üst sulu faz, oda sıcaklığında 1 dakika boyunca 16,000 x g&#39;de santrifüjleyin. </li><li> 1 dakika için 16,000 x g&#39;de kloroform / IAA, santrifüj, bir hacim ekleyin ve üst akıcı faz ekstrakte edin. </li><li> Adımı tekrarlayın 1.4.3. </li><li> Etanol 2 hacimleri ekleyin ve 1 saat ya da O / N -20 o C&#39;de bırakın. </li><li> 10 dakika boyunca 4 ° C&#39;de 16,000 x g&#39;de santrifüjleyin. süpernatant atın. </li><li> % 70 soğuk etanol ile pelet yıkanır ve TE içinde 2.5 ul başına approximatively 1 ug cDNA (10 mM Tris-HCI, 1 mM EDTA, pH 7.5) yeniden askıya. </li></ol></li><li> İki prob, digoxigenin (DIG) ile etiketlenmiş bir ve flöresin izotiyosiyanat (FITC) ile diğer sentez. <ol><li> Anti-se yapmak için uygun RNA polimeraz seçin(Hedefe mRNA tamamlayıcı) nse sonda. Bu örnek için, Aspa probu yapmak için T7 RNA polimerazını kullanır. </li><li> In vitro transkripsiyon reaksiyonu hazırlayınız: 1 linearize DNA ug, 4.0 ul 5 x transkripsiyon tamponu, 100 mM DTT, 6.0 ul 10x DIG ya da FITC RNA işaretleme karışımı içeren dNTP 2.0 ul, 1 ul RNaz inhibitörü ve 20 - Tr 40 RNA polimerazının birim; DEPC arıtılmış su ile 20 ul son hacmi oluşturuyor. </li><li> 1.5 saat 37 o C&#39;de inkübe edin. </li></ol></li><li> % 1.0 agaroz jeli üzerinde transkripsiyon reaksiyonunun IKAZ1 ul reaksiyon çalıştığı kontrol etmek için. Jel doğrusal şablonu ve prob parlak bir bant göstermesi gerekir. </li><li> melezleme tamponu ile 100 ul birimi oluşturacak ve -80 ° C&#39;de 10 ul alikotları saklayın. </li></ol> 2. perfüzyon, Fiksasyon ve Doku Koleksiyonu <ol><li> DİKKAT: (PFA) paraformaldehid taşırken hem katı hem deSulu, PPE giymek ve bir güvenlik kabini kullanın. ısı (55 ° C) PFA 1x olarak fosfat tamponlu tuzlu su (PBS) çözeltisi (ağ / hac)% 4 çözülmesiyle formaldehid çözeltisi hazırlayın. filtre kağıdı ile formaldehit çözüm Filtre. </li><li> damıtılmış su içinde sükroz (ağ / hac)% 20 eritilerek sukroz çözeltisi hazırlayın ve% 0.1 (h / h) DEPC çözeltisi ilave edilir. gevşek kapaklı ve otoklav ile oda sıcaklığında O / N bırakın. </li><li> Son aşamada pentobarbitonun intra-peritoneal enjeksiyonu (50 mg / kg) ile fare uyuşturan. ayak tutam anestezi derinliği değerlendirmek ve geri çekme refleksi yokluğu derin anestezi gösterir. </li><li> Fare derin anestezi altına alındıktan sonra, göğüs kafesine altında bir kesi yapmak ve her iki tarafta göğüs kafesinin kesti, kalp maruz kaldırın. </li><li> kalbin sol ventrikül içine 25 G iğne takın. kalbin sağ atrium küçük bir kesi yapmak. </li><li> 20 mi, PBS ile hayvan ve daha sonra 40 mi formaldehid çözeltisi serpmek. P30 ve üzerifareler, 12 ml / dk&#39;lık bir perfüzyon oranı kullanıldığında daha genç hayvanlar daha düşük bir oranı kullanım için (7-10 ml / dakika). </li><li> beyin teşrih. <ol><li> Kulağımıza gelen bir kesim posterior yaparak cerrahi makas ile fare kafasını Sever. deride bir kaudal orta hat kesi yapmak ve kafatası cilt kaldırmak için rostrally çalışır. </li><li> kaudal kısmından başlayarak, orta hat boyunca kafatasının üst kısmından ve İris makasla gözler arasındaki kesti. Bir kemik plaka bir tarafını her zaman devrilir ve cımbız ile kapalı yakalamaya tarafından parietal ve frontal kemik plakaları çıkarın. </li><li> Yavaşça cımbız ile ön kısmından yukarı doğru beyin tilt ve optik sinirleri ve diğer kranial sinirler kesilir. Yavaşça kafatası dışına beyin kaldırın. </li></ol></li><li> Bir fare koronal beyin matriks içine beyin yerleştirin. Bir tüy jilet ile 3 yaklaşık 4 mm parçalar halinde beyin dilimleyin. </li><li> % 4 PFA çözeltisi içine dilim aktarın ve 4 ° C&#39;de O / N inkübe edin. </li><li> aktarım beyindilim-DEPC işlenmiş% 20 sukroz çözeltisi ve 4 ° C &#39;de, O / N kuluçkalanması </li><li> kuru cryomould içine her beyin dilim koyun, optimum kesme sıcaklığı (OCT) orta ile çevreleyen ve kuru buz üzerinde dondurma. -80 ° C&#39;de saklayın. </li></ol> 3. Cryosectioning <ol><li> Bir kriyostat içinde donmuş doku 15 mikron bölümleri kesmek ve mikroskop lamı üzerine bölümleri toplamak. Eğer gerekirse, DEPC ile muamele edilmiş, PBS ile bölümleri ıslak ve ince bir fırça ile düzleştirin. </li><li> Doku slayt uygun olduğundan emin olmak için yaklaşık 1 saat boyunca kurumaya slaytlar bırakın. </li></ol> 4. Melezleme <ol><li> 65 ° C yıkama tamponu hazırlayın: 150 mM NaCI, 15 mM Na-3 Cı 3H 5 O (COO) 3 (= 1x tuzlu su, sodyum sitrat), 50% (h / h) formamid ile% 0.1 (h / h) Tween 20. </li><li> 100 mM maleik asit, 150 mM NaCI,% 0.1 (h / h) Tween-20, pH 7,5: MABT tamponu hazırlayın. </li><li> DIG etiketli ve FITC-işaretlenmiş (iki prob seyreltind) hibridizasyon tampon maddesi içinde 1 / 1.000 65 ° C&#39;ye önceden ısıtılmış ve iyice karıştırın. </li><li> Her bir slayt, coverslipwith fırında pişmiş (200 ° C) lamelleri üzerine hibritleme karışımının yaklaşık 300 ul uygulayın ve nemli bir oda içinde 65 ° C sıcaklıkta O / N inkübe edin. </li><li> önceden ısıtılmış yıkama tampon içeren bir Coplin kavanoza slaytlar aktarın. Yıkama tamponu ile 65 ° C &#39;de iki kez 30 dakika için slaytlar yıkayın. </li><li> Oda sıcaklığında, 10 dakika süreyle MABT ile üç kez slaytlar yıkayın. </li></ol> FITC Probe 5. Görselleştirme <ol><li> ISH engelleme tamponu hazırlayın: MABT% 2 bloke reaktif ve% 10 ısı ile inaktive edilmiş koyun serumu. </li><li> İsteğe bağlı: Gerekli antikor çözümleri hacmini azaltmak için slayt doku bölümleri etrafında daireler çizmek için bir hidrofobik kalem kullanın. </li><li> ISH nemlendirilmiş bir odada Blokaj tamponu ile oda sıcaklığında 1 saat için slaytlar inkübe edin. </li><li> bir yaban turbu peroksidaz ile 4 ° C&#39;de (POD) de slaytlar O / N inkübe - konjugatD, anti-FITC antikoru 1/500 (h / h) ISH engelleme tamponu seyreltilmiştir. </li><li> % 0.1 (h / h) Tween-20 (PBST) ile PBS içeren bir Coplin kavanoza slaytlar yerleştirin. her PBST içinde 10 dakika süre ile 3 kere yıkayın ve taze PBST ile değiştirin. </li><li> Kullanımdan hemen önce, Amplifikasyon seyreltici olarak 100 kez seyreltilmesi ile floresan tiramid hazırlar. Bu örnek için, FITC-tiramid kullanımı. </li><li> slaytlar bu ekleyin ve oda sıcaklığında 10 dakika bekletin. </li><li> Bir Coplin kavanoza slaytlar yerleştirin ve 10 dakika süre ile PBST içinde 3 kez yıkayın. </li></ol> DIG Probe 6. Görselleştirme <ol><li> 0.1 M Tris-HCl, pH 8.2 hazırlanır. </li><li> Oda sıcaklığında 1 saat için ISH bloke edici tampon ile slaytlar inkübe edin. </li><li> bir alkalin fosfataz (AP) ile 4 ° C&#39;de slaytlar O / N inkübe seyreltildi 1 / 1.500 (h / h) ISH engelleme tamponu, anti-DIG antikoru ile konjüge edilmiş. </li><li> 10 dakika için üç kez MABT ile yıkayınız. </li><li> Oda sıcaklığında 5 dakika boyunca 0.1 M Tris-HCI pH 8.2 ile iki kez yıkanır. </li><li>Kullanımdan hemen önce, 0.1 M Tris, pH 8.2 tabletler çözülmesiyle Hızlı Kırmızı solüsyon hazırlanır. 0.22 um&#39;lik bir filtreden filtre solüsyonu. </li><li> nemli bir oda içinde Hızlı Kırmızı çözelti ile 37 ° C&#39;de slaytlar inkübe edin. uygun gelişimi için geçen sürenin de, çökeltinin oluşumuna izlemek için flüoresan mikroskop ile düzenli slaytlar edin. 3 saat - Bu örnekte, 2, sonra reaksiyonu durdurun. </li><li> 10 dakika için üç kez PBST ile yıkanır. </li></ol> 7. İmmünhistokimya <ol><li> IHC bloke tamponu hazırlayın:% 10 PBST (birincil antikor konak için farklı türlerin) (h / h) serum. </li><li> IHC nemlendirilmiş bir odada, oda sıcaklığında 1 saat süre ile blokaj tamponu ile slaytlar inkübe edin. </li><li> Primer antikor hazırlanması (birincil antikor konakçıya farklı türlerin)% 5 serum ile PBST içinde, uygun bir seyreltme oranında primer antikor seyreltin. Bu örnekte, 1/400 (h / h) seyreltmede bir tavşan anti-olig2 antikor kullanımı. </li><li>4 ° de CO / N primer antikor çözeltisi içinde inkübe edin. </li><li> 10 dakika için üç kez PBST ile yıkanır. </li><li> İkincil antikor hazırlanması: Bu örnekte ve% 0.1 (h / h) Hoechst 33258. (farklı bir birincil antikor konak türlerin)% 5 ile PBST içinde, uygun bir seyreltme oranında (hac / hac), serum, bir florofor-konjuge sekonder antikor seyreltik 1 / 1.000 (h / h) seyreltmede ikincil antikor Alexa647 bir eşek anti-tavşan kullanın. </li><li> 1 saat boyunca oda sıcaklığında ikincil antikor çözeltisi içinde inkübe edin. </li><li> 10 dakika için üç kez PBST ile yıkanır. </li></ol> 8. Montaj <ol><li> Kısmen oda sıcaklığında slaytlar kuru ve, floresan montaj ortamına sahip monte edilir. Hızlı Red için 570 nm, FITC için 488 nm, olig2 immüno için 647 nm ve Hoechst için 350 nm: 10X altında konfokal mikroskop sonra, 20X ve aşağıdaki uyarma dalga boyları ile 4 farklı kanalları kullanarak 63X hedefleri görüntü kuru ve slaytlar bırakın . </li></ol>

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The authors declare that they have no competing financial interests.

Bu protokol immün tarafından takip In situ hibridizasyon çift RNA için bir adım-adım prosedürü sağlar. Biz Aspa birkaç beyin bölgelerindeki olgun oligodendrosit ifade edilmektedir onaylamak için bu protokolü kullanılır. Bu çok aşamalı prosedür hassasiyetini etkileyebilir ve kaçınılmalıdır pek çok potansiyel tuzaklar vardır. İlk olarak, transkripsiyon reaksiyonu için tüm çözümleri ve depolama tamponlar RNaz ücretsiz olması gerekir. İkinci o...

Merkezi sinir sisteminde (CNS) içinde en bol hücrelerdir glial hücreler, oligodendrositlerin (CNS myelinating hücreleri), oligodendrositler öncüleri (aynı zamanda &quot;NG2 hücreler&quot; olarak da bilinir OP), astrositler ve mikroglia içermektedir. Nörolojik hastalıklarda 1 glial hücreler ve bunların potansiyel rolleri fonksiyonları artan bir ilgi vardır. Örneğin, Canavan hastalığı (CD) genellikle yaş 2,3 10 yıl önce ölüme yol açan, erken süngersi leukodystrophy ve nöronların ilerleyici kaybı ile bebeklik döneminde başlayan bir kalıtsal nörodejeneratif bir hastalıktır. Yol Aspartoacyclase (ASPA&#39;nın) genindeki mutasyonlar ölçüde CD ASPA&#39;nın aktivitesi 4 düşürülen tespit edilmiştir. ASPA N-asetilaspartat (NAA) deasetilasyonu katalize bir enzim, yüksek beyin içinde konsantre molekülü üreten asetat, aspartat 5-7. Birçok CD hasta nedeniyle ASPA ac eksikliği NAA daha yüksek olduğunu göstermektedirtivity. Bazı çalışmalar NAA türetilmiş asetat gelişimi sırasında yağ asitleri / beyinde lipidlerin önemli bir kaynağı olabilir ve 3,5,6 aşağı kırılması NAA yetmezliği nedeniyle gelişimi sırasında miyelin sentezi azaldığı CD neden olabilir spekülasyon. ASPA ağırlıklı beyin böbrek, karaciğer ve beyaz cevherde bulunan ve CD&#39;deki ASPA önemli rolü verilir, beyindeki bu enzimin hücre ifadesi birkaç laboratuvarlar tarafından incelenmiştir. Beyinde ASPA enzimatik aktivite bakarak, daha önceki çalışmalarda beyin gelişimi sırasında ASPA aktivite artışı miyelinasyon 8-10 zaman sürecini paralellik bulundu. Hücresel düzeyde, enzimatik aktivitesi için hem de in situ hibridizasyon (ISH) ve immünohistokimya (IHC) deneyleri ASPA&#39;nın esas olarak beyinde konumlandığı, fakat nöronlar veya astrosit 11-16 oligodentrositler ifade olduğunu göstermektedir analizleri. Birkaç çalışmalarda ASPA da olabilir oCNS 12,14 olarak mikroglia olarak ifade edilebilir. Operasyonel olarak ASPA ifadesine Şimdiye kadar veriler sınırlıdır. Nöronlar, astrositler, OP&#39;lerle, yeni kurulan oligodendrositler, myelinating oligodendrositler, mikroglia, endotelyal hücreler ve perisitlere dahil fare serebral korteks farklı hücre tiplerinin transcriptomes RNA sıralanması 17 ile analiz edildi Yeni bir araştırmaya göre, ASPA sadece oligodendrositlere ifade edilir , oligodendrositlerin myelinating özellikle (http://web.stanford.edu/group/barres_lab/brain_rnaseq.html). Beyinde ASPA ifade deseni bu çalışmalara rağmen, belirsizliklerin bir dizi kalır. Farklı teknikler gen ekspresyonu incelemek için kullanılabilir. IHC doku kesitlerinde bir gen ekspresyonunun işlevsel bir ürün (örneğin, protein) tespit etmek için yaygın olarak kullanılan bir yöntemdir. uygulama ve özgünlüğü tabi t gibi onun büyük yarar rağmen, bu tekniğin sınırlamaları vardırihtiyaç duyulan antikorun mevcudiyeti ve özgünlüğü o. Buna karşılık, ISH mRNA düzeyinde bir genin ekspresyonunu ortaya çıkarmak mümkün olma avantajına sahiptir. Bununla birlikte, özel hücre tipleri için bir gen ekspresyonunu lokalize etmek için aynı anda birden fazla prob kullanımı teknik olarak zor olabilir. Bu yazıda, bir proteinin floresan immüno ile in situ hibridizasyon çift floresan RNA birleştiren bir protokol açıklar. Biz fare beyninde Aspa salgılanma modelini incelemek için teknikler bu dizi kullandık. Bu yöntem, konfokal mikroskopi kullanılarak gen ekspresyonunun hassas çalışma sağlar.

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combining double fluorescence in situ hybridization with immunolabelling for detection of the expression of three genes in mouse brain sections

Beyin hücreleri, örneğin nöronlar, astrositler, oligodendrositler, oligodendrosit öncüleri ve mikroglia farklı gen ifadesinin saptanması, immün spesifik primer ya da sekonder antikorların yokluğuyla engellenebilir. Burada üçüncü geni tarafından kodlanan proteine ​​karşı yöneltilmiş yüksek özgüllük bir antikor ile immün ardından, iki gen spesifik problar ile in situ hibridizasyon, çift floresan kullanılarak aynı beyin kısmında üç farklı gen ekspresyonunu tespit etmek için bir protokol açıklar. Aspartoacyclase (ASPA) geni, mutasyonlar nadir insan beyaz madde hastalığı neden olabilir - Canavan hastalığı - oligodendrositler ve mikroglia ancak astrositler ve nöron ifade edilmesi düşünülmektedir. Bununla birlikte, beyinde ASPA kesin ifade deseni henüz belirlenmemiştir zorundadır. Bu protokol ASPA olgun oligodendrosit a bir alt kümesi olarak ifade edildiğini belirlemek için bize izin verdiND genellikle gen ekspresyon paterni çalışmalar çeşitli uygulanabilir.

Bu makalede, bir fare beyin bölümlerinde immüno ardından çift flüoresan ISH için bir yöntem tarif eder. Bu protokolün kısa bir açıklama ilk adım Aspa ve MBP (miyelin bazik proteini) özel problar sentezlemek oldu Şekil 1&#39;de gösterilmiştir. Probes sentezlenmiş olduğunu kontrol etmek için, her bir reaksiyonun küçük bir kısım, bir agaroz jel üzerinde yürütülmüştür. Hafif lineer şablonu ve RNA probu büyük miktarda <stro...

Watch this Scientific Journal Video about Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections at JoVE.com

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

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

Çift Floresan birleştiren<em&gt; In Situ</em&gt; Fare Beyin Bölüm Üç Genlerin İfade tespiti için immünoişaretleme ile Hibridizasyon

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

combining-double-fluorescence-situ-hybridization-with-immunolabelling

Research

JoVE Journal

Neuroscience

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

13.0K Görüntüleme.  University College London. Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

Çift Floresans In situ Taze Beyin Bölüm Hibridizasyon

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

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

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

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

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

Lipofilik boya birleştiren In situ Hibridizasyon, İmmünohistokimya ve Histoloji

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

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge in the field of virology; in particular for nuclear-replicating persistent DNA viruses that involve animal models for their study. Herpes simplex virus type 1 (HSV-1) establishes a life-long latent infection in peripheral neurons. Latent virus serves as reservoir, from which it reactivates and induces a new herpetic episode. The cell biology of HSV-1 latency remains poorly understood, in part due to the lack of methods to detect HSV-1 genomes in situ in animal models. We describe a DNA-fluorescent in situ hybridization (FISH) approach efficiently detecting low-copy viral genomes within sections of neuronal tissues from infected animal models. The method relies on heat-based antigen unmasking, and directly labeled home-made DNA probes, or commercially available probes. We developed a triple staining approach, combining DNA-FISH with RNA-FISH&#160;and immunofluorescence, using peroxidase based signal amplification to accommodate each staining requirement. A major improvement is the ability to obtain, within 10 &#181;m tissue sections, low-background signals that can be imaged at high resolution by confocal microscopy and wide-field conventional epifluorescence. Additionally, the triple staining worked with a wide range of antibodies directed against cellular and viral proteins. The complete protocol takes 2.5 days to accommodate antibody and probe penetration within the tissue.

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

We established a fluorescent in situ hybridization protocol for the detection of a persistent DNA virus genome within tissue sections of animal models. This protocol enables studying infection process by codetection of the viral genome, its RNA products, and viral or cellular proteins within single cells.

Floresan Nöronal Dokularda Kalıcı DNA Virüs Genomu ve Transkriptlerinin Algılama In situ Melezleme immün ile birlikte

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

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

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

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

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

Sonradan wholemount için komple Omurilik Yaralanmalı ve Beyin Diseksiyon Protokolü<em&gt; Yerinde</em&gt; Larva Deniz taşemen içinde Hibritleşme

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

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

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

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

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

Yüksek Çözünürlük kullanma Beyin bölümlerde Axonally Lokalize mRNA&#39;larının Algılama<em&gt; In Situ</em&gt; Melezleme

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

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

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

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

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

Çabuk<em&gt; In Situ</emSerotonin Sendromu ile Ratlarda Paraformaldehyde-öneki Beyin oligonükleotid Problar kullanarak&gt; Melezleme

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

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

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

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

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

Locust Antenlerinde Odorant Reseptör Genlerinin RNA ile Lokalizasyonu<em&gt; Yerinde</em&gt; Hibridizasyon

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

Large-scale Three-dimensional Imaging of Cellular Organization in the Mouse Neocortex

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical properties, synaptic connections, and in vivo functions, but their basic functional and anatomical organization from cellular to network scale is poorly understood. Here we describe a method for the three-dimensional imaging of fluorescently-labeled neurons across large areas of the brain for the investigation of the cortical cellular organization. Specific types of neurons are labeled by the injection of fluorescent retrograde neuronal tracers or expression of fluorescent proteins in transgenic mice. Block brain samples, e.g., a hemisphere, are prepared after fixation, made transparent with tissue clearing methods, and subjected to fluorescent immunolabeling of the specific cell types. Large areas are scanned using confocal or two-photon microscopes equipped with large working distance objectives and motorized stages. This method can resolve the periodic organization of the cell type-specific microcolumn functional modules in the mouse neocortex. The procedure can be useful for the study of three-dimensional cellular architecture in the diverse brain areas and other complex tissues.

Here we describe a procedure for tissue clearing, fluorescent labeling, and large-scale imaging of mouse brain tissue which, thereby, enables visualization of the three-dimensional organization of cell types in the neocortex.

Hücresel Örgütü fare yeni korteksimiz yılında büyük ölçekli üç boyutlu görüntüleme

The mammalian neocortex is composed of many types of excitatory and inhibitory neurons, each with specific electrophysiological and biochemical ...

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

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

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

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

Multipleks Floresan In Situ Hibridizasyonun Floresan İmmünohistokimya ile Taze Dondurulmuş veya Sabit Fare Beyin Kesitlerinde Birleştirilmesi

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

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting the expression of G protein-coupled receptors (GPCRs). Formalin-fixed jugular-nodose ganglia were processed with the RNAscope technology to simultaneously detect the expression of two representative GPCRs (cholecystokinin and ghrelin receptors) in combination with one marker gene of either nodose (paired-like homeobox 2b, Phox2b) or jugular afferent neurons (PR domain zinc finger protein 12, Prdm12). Labeled ganglia were imaged using confocal microscopy to determine the distribution and expression patterns of the aforementioned transcripts. Briefly, Phox2b afferent neurons were found to abundantly express the cholecystokinin receptor (Cck1r) but not the ghrelin receptor (Ghsr). A small subset of Prdm12 afferent neurons was also found to express Ghsr and/or Cck1r. Potential technical caveats in the design, processing, and interpretation of multiplex ISH are discussed. The approach described in this article may help scientists in generating accurate maps of the transcriptional profiles of vagal afferent neurons.

Detection of G Protein-coupled Receptor Expression in Mouse Vagal Afferent Neurons using Multiplex In Situ Hybridization

Multiplex in situ hybridization (ISH) was employed to simultaneously visualize the transcripts for two G protein-coupled receptors and one transcription factor in the entire vagal ganglionic complex of the adult mouse. This protocol could be used to generate accurate maps of the transcriptional profiles of vagal afferent neurons.

Multipleks In Situ Hibridizasyonu Kullanan Fare Vagal Afferent Nöronlarında G Protein Bağlantılı Reseptör ekspresyonunun tespiti

This study describes a protocol for the multiplex in situ hybridization (ISH) of the mouse jugular-nodose ganglia, with a particular emphasis on detecting ...