NIH / NIDCD ve RP Schmitt Vakfı hibe tarafından desteklenen çalışın.

Bu protokol, önce bir ya da iki beyin dokusu 1-7 transkript türlerini tespit etmek için, biz ve diğerleri tarafından geliştirilen standart radyoaktif ve radyoaktif olmayan in-situ hibridizasyon yöntemleri, dayalı geliştirilen ve rafine oldu. Aşağıda açıklanan protokol, son kullanıcı tarafından seçilen usul kesintilerinin sayısına bağlı olarak 2 veya 3 gün toplam uzunluğu vardır. Aşağıda ayrıntılı olarak tüm adımları Spastine Özgü Riboprop hibridizasyon adım ve sonrası hibridizasyon yıkar dışında, oda sıcaklığında yapılacak. Bu yöntemde ilgili tüm adımları için gerekli çözümleri ve tamponlar bu protokol sonunda bulunabilir. 1. Doku hazırlanması ve Kesit <ol><li> Başını kesmek ve deneğin beyin hızla ayıklamak ve yeterli büyüklükte plastik bir kalıp içine yerleştirin. </li><li> Kapak Doku Tek gömme orta beyin ve hızlı bir şekilde hızlı dondurma dry-ice/alcohol banyo plastik kalıp. Dondurulmuş doku -80 ° C kullanana kadar saklanabilir. </li><li> Bir Kriyostat kullanarak, tahsil SuperFrost Plus Slaytlarınıza slayt başına 2 veya 3 beyin bölümlerde toplamak. Bölümlerde kalınlığı 10-12 mikron olmalıdır. Slaytlar -80 ° C kullanana kadar saklanabilir. </li></ol> 2. Probe Arıtma Sephadex G50 Sütunlar hazırlanması &quot;Sephadex sütunları ticari kaynaklardan satın alınabilir, ancak, sütunlar, prob arıtma için gerekli olacak nesil için aşağıdaki düşük maliyetli bir alternatif sağlar. <ol><li> RNaz-free, oda sıcaklığında DEPC ile tedavi edilen su (örneğin, 2 gram toz, DEPC arıtılmış su 100 ml), mix çözüm kısaca ve mağaza Sephadex G50 toz Hidrat yeterli miktarda aşırı Sephadex G50 hızlandırabilir. </li><li> Sephadex G50 yağış (suyun üst tabakası) süpernatantı. </li><li> 3 - 5 kat yukarıdaki işlemi tekrarlayın. </li><li> Son yıkamadan sonra, kullanana kadar 4 Sephadex G50 TE tamponu (1:1 oranı) çözüm ve mağaza ° C yeniden askıya. </li><li> 1 ml steril bir şırınga içine yerleştirin cam yünü otoklavlanabilir ve kompakt bir alt katmanı yapmak için piston ile sıkıştırmak ve sonra 15 ml Falcon tüp içine şırınga. </li><li> TE Sephadex G50 çözümü karıştırın. Bu çözüm ile şırınga / sütun doldurun. </li><li> 1000 rpm&#39;de 30 saniye süreyle sütun santrifüjleyin. </li><li> Sütun neredeyse tamamen Sephadex G50 boncuklar ile dolana kadar bu işlemi tekrarlayın. </li><li> Sütun sütun yıkama tamponu 200 ul uygulayın ve 1000 rpm&#39;de 2 dk santrifüj. Flow-through atın. </li><li> 200 ul sütun sütun engelleme tampon uygulayın ve 1000 rpm&#39;de 2 dakika döndürün. Sütun dengelenmesi için bu adımı 4-5 kez tekrarlayın. ° C kullanana kadar Parafilm ile mühürlenmiş Sütunlar 4 saklanabilir. </li></ol> 3. Riboprobes Etiketlenmesi ve Arıtma Detaylı, tek bir Spastine Özgü Riboprop üretim ve arıtma Aşağıda. DFISH için, her sonda hazırlık probların biotin etiketli UTP ile diğeri ise digoxigenin (DİJİTAL) etiketli UTP etiketli olacağını dışında, aynı metodoloji içerecektir. <ol><li> Duygusu (kontrol) veya antisens riboprobes oluşturmak için bir ilgi konsantre (&gt; 150 ng / ml) ve saflaştırılmış Lineerleştirilmiş cDNA çözüm hazırlayın. </li><li> 1.5 ml mikrofuge&#39;de tüp, cDNA şablon, 5X prob etiketleme tampon 2 ul, 10X DIG 1 ul (veya biotin) etiketleme karışımı, RNasin 0.5 ul ve uygun RNA polimeraz 1 ul saflaştırılmış 0.5-1 mg eklemek ve son hacmi RNAse-free (DEPC tedavi) su ile 10 ul çözüm getirmek. </li><li> 37, bir su banyosu içinde çözüm ° C de 2 saat süreyle inkübe edin. </li><li> Çözüm ve sütun engelleme tampon 39 ul; tRNA 1 ul (20 mg / ml hisse senedi) ekleyin. </li><li> Prob arıtma Sephadex G50 sütun hazırlayın. Bu amaçla 50 ul engelleme tampon, sütun ekleyin ve 1000 rpm&#39;de 10 saniye boyunca döndürün. Kolon (yani tam 50 ul sütuna uygulanan santrifüj döngüsü sonra kurtarıldı.) Dengelenmesi için bu adımı 2-3 kez tekrarlayın. </li><li> Sephadex G50 sütun, pozisyon, sütunun alt kısmında yeni bir mikrofuge&#39;de tüp prob çözümü uygulayın ve saflaştırılmış 50 ul Spastine Özgü Riboprop çözüm elde etmek için 1000 rpm&#39;de 3 dakika döndürün. </li><li> Ya bir standart formaldehit agaroz RNA jel, ya da bir spektrofotometre kullanarak etiketli Spastine Özgü Riboprop kalite ve verim değerlendirin. </li></ol> 4. Post-fiksasyon, Asetilasyon ve Hibritleşme <ol><li> -80 ° C derin dondurucu bölümleri çıkartın ve oda sıcaklığına denge sağlaması için izin. Daha sonra, 5 dakika süreyle soğuk, taze% 3 paraformaldehid çözüm bölümler kuluçkaya yatmaktadır. </li><li> Kısaca p bölümleri durulamahosphate iki kez tamponlu salin (PBS). </li><li> Standart bir alkol serisi (70, 95, ve% 100, 2 dk) ile bölümleri, kurutmak ve hava kurumaya bırakın. </li><li> 10 dakika bir asetilasyonu çözüm bölümleri inkübe edin. </li><li> 2X SSPE bölümler 3 kez durulayın. </li><li> Yukarıda açıklanan standart alkol serisi ile tekrar bölümleri dehydrate ve onları kurumasını sağlar. </li><li> Hibridizasyon çözüm yeterli bir hacmi hazırlayın ve hem riboprobes çözüm (prob konsantrasyonu her probu için 1 ng / ml) ekleyin. Hibridizasyon çözüm toplam hacmi (hibridizasyon çözüm Bölüm başına 16 ul) melezleşmiştir bölümlerin sayısına göre belirlenir. </li><li> Herhangi bir hava kabarcıkları bindirme sağlayarak doku doku ve kapak slaytlara hibridizasyon çözüm yeterli hacim uygulayın. </li><li> Metal bir slayt tutucuya yerleştirin slaytlar. Daldırın tutucu bir mineral yağ banyosu içinde 65 ° C gece ayarlayın. Bu lamelleri (yani, slayt sahibinin lateral mineral yağ kabın alt kısmına temas edilmelidir) yukarı bakacak olun. </li></ol> 5. Post-Hibridizasyon yıkar <ol><li> Ertesi gün, mineral yağ banyosu dikkatle slayt tutucu kaldırmak ve slaytlar kısaca aşırı yağ kaldırmak için kloroform durulayın. </li><li> Yer 2X SSPE çözümü için bir 5-10 dakika slaytlar. Lameller çözelti içinde iken slaytlar ayırmak gerekir. </li><li> Yeni bir 2X SSPE çözüm slaytları transferi ve 1 saat oda sıcaklığında saklayın. </li><li> 2X SSPE artı% 50 formamid içeren bir çözüm bölümleri aktarın. Bu çözümün sıcaklık gecelik hibridizasyon yöntemi kullanılan sıcaklık uyumlu olmalıdır. 1.5 saat süreyle bu çözüm slaytlar tutun. </li><li> 0.1x SSPE çözüm hibridizasyon adım aynı sıcaklıkta önceden ısıtılmış bölümler aktarın. 30 dakika için bu çözüm bölümleri inkübe edin. Ek bir 30 dakika için bu adımı tekrarlayın. </li></ol> 6. Riboprobes Algılama ve Görselleştirme <ol><li> % 0.3 &#39;lük hidrojen peroksit 10 dakika eklenmiştir TNT tampon Slaytları aktarın. </li><li> TNT tampon, 3 kez (10 dk) bölümleri yıkayın. </li><li> Bir DAKO kalem kullanarak, beynin bölümleri içeren alanı çevresinde iyi çizin. Daha da önemlisi, dikkatli bölümlere kuru değil sağlamak için dikkatli olunmalıdır. </li><li> Her bir slayt 150 ul TNB tampon uygulayın ve nemli bir odasında, bu çözüm bölümleri 30 dakika inkübe edin. </li><li> Devrilir slaytlar aşırı TNB çözüm çıkarın. </li><li> TNB çözüm 2 saat süreyle nemli odasında peroksidaz anti-DIG-konjuge antikor, ve mağaza slaytlar içeren 150 ul uygulayın. Antikor konsantrasyonu önce dFISH yürüten ilgi her transkript için ayrı ayrı tespit edilmelidir. </li><li> TNT tampon bölümleri yıkayın; 3 kez, her biri 10 dk. </li><li> Alexa her slayt için 594-konjuge tyramide çalışma çözeltisi 150 ul uygulayın ve 1 saat için nemli bir oda saklayabilirsiniz. Bu çözüm üretici yöne göre hazırlanmalıdır. </li><li> TNT tampon bölümleri yıkayın; 3 kez, her biri 10 dk. </li><li> - 30 dakika inkübe bölümleri TNT tampon hangi% 0.3 &#39;lük hidrojen peroksit 10 eklendi olmuştur. </li><li> TNT tampon bölümleri yıkayın; 3 kez, her biri 10 dk. </li><li> Slayt başına 150 ul TNB tampon uygulayın ve 30 dakika için nemli bir oda tutmak. </li><li> Devrilir slaytlar aşırı TNB çözüm çıkarın. </li><li> TNB çözüm 2 saat süreyle nemli odasında peroksidaz-konjuge anti-biotin antikor, ve mağaza slaytlar içeren 150 ul ekleyin. </li><li> TNT tampon bölümleri yıkayın; 3 kez, her biri 10 dk. </li><li> Bir Alexa 150 ul 488-konjuge slaytlar ve 1 saat süreyle inkübe bölümleri tyramide çalışma çözümü uygulayın. Bu çözüm, üreticinin tavsiyelerine göre hazırlanmalıdır. </li><li> TNT tampon bölümleri yıkayın; 3 kez, her biri 10 dk. </li><li> Hoechst çözümü (TNT tampon 1:1000) 150 ul bölümlere ekleyin ve 2 dakika süreyle nemli odasına saklayın. </li><li> TNT tampon bölümlerin yıkayın; 3 kez, her biri 5 dakika. </li><li> Lamel bir floresan uyumlu montaj orta (örneğin, Vectashield veya antifade uzatmak) bölümleri. </li></ol> 7. Temsilcisi Sonuçlar <img src="/files/ftp_upload/2102/2102fig1.jpg" alt="Şekil 1" /> Şekil 1: Biz burada zebra ispinozu beyin elde temsilcisi dFISH sonuçlarını gösterir. Caudomedial nidopallium (NCM), memeli işitsel korteks Songbird analog elde fotomikrografı gösterilir. Beyin bölümleri parvalbumin (A), inhibitör nöronların alt nüfus için bir marker yöneltilen bir biotinlenmiş Spastine Özgü Riboprop melezleşmiştir ve Ribo DIG etiketliprob, şarkı odaklı nöronlar için güvenilir bir belirteç aktivite bağımlı gen zenk (B) karşı yöneltilmiş. C) (A) ve (B) işitsel deneyim ile aktive olan inhibitör nöronların bir nüfus gösterir Yerleşimi. Oklar ve ok uçları, her iki riboprobes özel etiketli hücreleri gösterir ve yıldızlarla ortak ilgi her iki transkript ifade temsilcisi nöronlar göstermektedir. Ölçek çubuğu = 25 mm.

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

Biz omurgalı beyin neurochemically ve işlevsel nasıl düzenlendiğini incelemek ve davranışsal ilgili duyusal uyaranlar etkisi, yetişkin beyin 8-10 nöronları genomik makine belirlemek için bu protokolü kullanılır . Biz başarılı, fare, sıçan ve ötücü beyin dokusu bu yöntem kullanılabilir, ancak bu protokol omurgalı türlerin bir dizi ve belki de, non-nöral dokuların elde edilen beyin bölümlerine kolayca adapte olacağını tahmin ediyoruz. Güvenilir sonuçlar ve en iyi sinyal elde e...

Çözümler <ol><li> Sütun yıkama tamponu: 50 ml DEPC arıtılmış su 10 mM Tris-HCl, 0.15 M NaCl, 0.05 mM EDTA, 50 mg / ml tRNA,% 0.1 SDS. </li><li> Sütun engelleme tampon: 50 ml DEPC arıtılmış su 10 mM Tris-HCl, 50 mM NaCl, 0.1 mM EDTA. </li><li> TNT tampon: 60 ml 1 M Tris-HCl, 5 M NaCl 18 ml ve 1.8 ml, DEPC arıtılmış su 600 ml X-100 Triton. </li><li> TNB tampon: 100 mM Tris-HCl, 8.3 mikrogram / ul BSA, 0.15 M NaCl ve% 3 Triton DEPC arıtılmış su X-100. </li><li> TE tampon: 10 mM Tris-HCl, pH 7.5 artı 1 mM EDTA, pH 7.5. </li><li> Asetilasyon çözüm: trietanolamin artı 0.5 ml, DEPC arıtılmış su 200 ml asetik anhidrit 2.7 ml. </li><li> Hibridizasyon çözüm:% 50 formamid, 2X SSPE, 2 mg / ul tRNA, 1 mcg / ml BSA ve 1 mg / ul DEPC arıtılmış su bir poli. Bölüm başına 16 l ihtiyacı var. </li></ol>

double fluorescence in situ hybridization in fresh brain sections

Burada bir çift floresan değiştirilmiş bir versiyonu tarif<em&gt; In situ</em&gt; Hibridizasyon (dFISH) yöntemi, taze donmuş beyin bölümlerde ilgi iki mRNA&#39;ların tespit etmek için optimize edilmiş. Grubumuz bu yaklaşımın başarılı gen co-yönetmelik çalışma kullandı. Daha spesifik olarak, tek bir hücre çözünürlükte, anatomik organizasyonu, nöro-kimyasal özellikleri, ve duyusal deneyim merkezi duyusal devreleri etkisini keşfetmek için, bu dFISH yöntem kullanılır. Bu protokol, fare, sıçan ve ötücü beyin dokusu onaylandı ancak kolayca diğer omurgalı türlerinin yanı sıra uyarlanabilir, non-nöral dokuların bir dizi olması bekleniyor. Bu filmde bu prosedürün ana adımları ayrıntılı bir gösteri sağlar.

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

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Bu protokol bir radyoaktif olmayan içerir<em&gt; In-situ</em&gt; Hibridizasyon işlemi, tek bir hücrenin çözünürlükte, omurgalı beynin ince bölümlerde aynı anda iki transkript türlerin belirlenmesini sağlar.

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 ...

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

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JoVE Journal

Neuroscience

Double Fluorescence in situ Hybridization in Fresh Brain Sections

16.0K Görüntüleme.  University of Rochester. Bu protokol bir radyoaktif olmayan içerir In-situ Hibridizasyon işlemi, tek bir hücrenin çözünürlükte, omurgalı beynin ince bölümlerde aynı anda iki transkript türlerin belirlenmesini sağlar.

Video: Double Fluorescence in situ Hybridization in Fresh Brain Sections

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

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

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

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

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 ...

Nörobilim

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

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

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

Küçük Hayvanlar Kronik Sinir Kayıtlar için Ultra-hafif Motorlu Microdrive Tasarımı ve Montajı

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

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

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

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

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

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 ...

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

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

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

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

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 ...

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 ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Yalıtım taze insan beyin dokusundan beyin kapiller

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

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 ...

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

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

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