This work was funded by the Deutsche Forschungsgemeinschaft DFG (Exc257, SFB958).

NOT: Tüm hayvan prosedürleri hayvanların herhangi bir gereksiz acı kaçınarak Alman Hayvan Bakım yasalarına uygun olarak Charité veya Üniversite Kliniği Jena ele alındı. 1. hazırlanması Çözümler ve Diseksiyon İşyeri <ol><li> Çözümler NOT: epitel diseksiyonu başlamadan önce aşağıdaki çözümleri hazırlayın. <ol><li> Diseksiyon işlemi için Çözümler: <ol><li> 140 mM NaCI, 5 mM KCI, 10 mM HEPES, 2 mM CaCI2, 1 mM MgCI2 ve 10 mM glukoz konsantrasyonları ile Ringer çözeltisi (pH 7.4) içinde hazırlayın. </li><li> - / - 2.68 mM KCI, 1.47 mM KH 2 PO 4, 136 mM NaCI, 8.1 mM Na-2 HPO 4 konsantrasyonlar çözeltisi (pH 7.4) içinde bir PBS hazırlayın. </li><li> - / -, 0.2 mM CaCI2,% 4 sukroz ve% 4 paraformaldehid 1x PBS konsantrasyonları ile bir tespit çözeltisi (pH 7.2) ile hazırlayın. Sabitleştirici çözelti içinde sukroz kriyo artırırkesit ve pick-up cryosections arasında. -20 ° C&#39;de saklayın. </li></ol></li><li> Boyama işlemi için çözümler <ol><li> 1x PBS konsantrasyonları ile PBS + / + çözeltisi hazırlayın - / -, 0.48 mM MgCl2, 0.9 mM CaCl2. </li><li> 1x PBS + / + ve% 0.1 Triton X-100 konsantrasyonları ile, PBST + / + çözelti hazırlayın. </li><li> 1x PBST ile + / + ve% 1 jelatin konsantrasyonları ile bir blokaj çözeltisi hazırlayın. </li></ol></li></ol></li><li> Işyeri <ol><li> Diseksiyon işyeri için, parlak aydınlatma ile bir diseksiyon mikroskobu, yanı sıra sıvı-bloker kalem kullanın. </li><li> Ringer çözeltisi, ve yapışkan cam lamlar ile doldurulmuş bir petri kabı, hazırlanır. </li><li> Aşağıdaki cerrahi aletler elde: cerrahi makas keskin ve bir künt ucu, düz uç şekli ile yaylı makas, ince kavisli ucu şekli ve bir jilet ile iki forseps bir çift (olan <strong&gt; Şekil 1A). </li></ol></li></ol> Burun Septumu 2. Hazırlık <ol><li> Gıda ve su ve uygun gündüz / gece döngüsü düzenli erişimi onaylı kafeslerde ev hayvanları. </li><li> Arka ayak refleksleri test ederek% 100 izofluran ve monitör analjezi ile ıslatılmış bir kapalı hazne içeren gazlı bez anestezi gerçekleştirin. Servikal dislokasyon burun boşlukları kan neden olabildiğinden, doğrudan anestezi uygulanmış farelerin başını kesmek. </li><li> Tüm kafatası ve burun kemiği açığa çıkarmak için deri kaldırmak ve iyice bir kağıt havlu kullanarak kalan kan ve doku silin. Alt çene ve ön dişleri çıkarın. </li><li> Maksilla (yanal) den septum (medial) bir tarafını ayırmak için sütür hattına 1-2 mm mesafe paralel bilateral burun dorsal kemik İnsizyon. Tek bir kesim ile burun bölün. Kemik kalıntıları ve konka hala septum takılı ise, dikkatlice çıkarın tamamen septum maruzdokunmadan. </li><li> Septum ve burun kemiği arasında, septum dorsal kenarı boyunca bir forseps ince kavisli ucu kaydırarak dorsal nazal kemiği çıkarın. , Kemik üzerinde hafif basınç uygulayın yukarı itin ve çıkarın. </li><li> Her bir oyuğun iki septum dokulara erişime bir sağlamak için dikkatli bir şekilde baş diğer tarafında maksilla çıkarın. Büyük hayvanlar (&gt; 28 P) hazırlanırken, koku ampuller kapsayan frontal kemik ucu çıkarın. </li><li> Onun hafif sarı renk (Şekil 1B) tarafından koku epiteli belirleyin. sınırındaki solunum epiteli beyaz ve mikroskop altında görülebilir hareketli kirpikler hareketini gösterir. İnce bahar makasla ile koku ve solunum epiteli arasındaki sınır boyunca kesin. </li><li> Kesim uzatın ve vomer kemik ventral bağlantıdan septum ayırın. Sonra septum ve Os etmoidit lamina cribrosa arasındaki sınır boyunca kesilmişör. septum artık tamamen kafasına bütün bağlantıları izole edilir. Şekil 1C gösterilen kesme konumlarını kullanın. </li></ol> Immün için Koku Epitelinin 3. İzolasyonu <ol><li> Ringer çözeltisi ile doldurulmuş bir petri yapışkan bir cam slayt yerleştirin. Camın bir yarım Ringer çözeltisi (Şekil 1A) &#39;de yer alan şekilde, bir petri kenarına yerleştirin. </li><li> Dikkatle petri koku epiteli ile etmoid kemik aktarmak için bir forseps kullanın. Forseps ipuçları ile kapma olmadan kaldırın. </li><li> Bir forseps ile etmoid kemiğin dik plaka tut ve dikkatli septum bir taraftan epitel kaldırmak için ikinci bir birini kullanın. Epitel kalkmasına dik plaka ve epiteli arasındaki kavisli ucu kaydırın. </li><li> Epitel Ringer soluti çevirir durumunda, siliyer tarafını belirlemek her zaman emin olunüzerine. Epitel çevirir ise, sonradan siliyer tarafı belirlemek pek mümkün değildir. Çoğunlukla, epitel içindeki silier yüzeyi ile rulo. </li><li> Şekil 1C gösterilen kesme pozisyonları ile kayıtlı doku şeklini karşılaştırın. Sınırda bir pozisyonda epitel tut ve cam slayt üzerine çekin. Doku lezyonları önlemek için forseps ile siliyer tarafına dokunmayın. </li><li> Septum çevirin ve diğer taraftan epitel ile işlemi tekrarlayın. </li><li> Bir kağıt havlu ile koku epiteli iki adet etrafında cam slayt kurulayın ve bir sıvı-bloker kalemle doku çevreler. </li><li> Oda sıcaklığında 10 dakika boyunca 150 ul sabitleyici çözeltisi ile doku sabitleyin. Kirpikler stabilite ve bütünlüğü ile ilgili olarak, test edilen antikorların çeşitli kısa sabitleme kez kullanmak. Bu 10 dakika kirpikler içinde sabit, ama muhtemelen bütün epitel dokusu vardır. Bu durumda, hemen s sonra mikroskop ile görselprosedürü yükümlüsünüzdür. Ancak, sabitleyici katı bireysel antikorlar için değişiklik gösterebilir. </li></ol> 4. Boyama Protokolü NOT: koku alma duyu nöronlarının siliyer yapıları korumak için çok dikkatli doku tutun. Pipetleme çözüm çıkarın. Mekanik kuvvetler ince kirpikler bozabilir gibi, epiteli üzerine doğrudan herhangi bir çözüm düşürmeyin. Başka türlü belirtilmediği sürece, oda sıcaklığında bütün adımları. <ol><li> Sabitleştirici çözüm çıkarın ve PBS + / + 2 kez yıkayın. </li><li> Bloke etme çözeltisi ile en az 1 saat süre ile epitel inkübe edin. </li><li> Herhangi bir çökelti kaldırmak için tam hızda 5 dakika boyunca (bu çalışmada kullanılan antikorlar için 200 1) ve santrifüj çözeltisi bloke antikor eritilmesi ile primer antikor çözeltisi hazırlayın. </li><li> 4 ° CO / K bir nemli oda içinde antikor çözeltisi ile epitel inkübe edin. </li><li> PBS + / + için 5 dakika ile yıkayın epitel en az 3 kez. </li><li>Tam hızda 5 dakika boyunca ve santrifüj: çözeltisi (1: 500) bloke antikor eritilmesi ile ikincil antikor çözeltisi hazırlayın. </li><li> Bir karanlık oda içinde 1 saat süre ile ikinci antikor çözeltisi ile epitel inkübe edin. </li><li> PBS + / + için 5 dakika ile yıkayın epitel en az 3 kez. </li><li> Bir jilet veya kağıt havlu ile sıvı-bloker çıkarın. 5 saniye süreyle damıtılmış su ile epitel yıkayın. </li><li> Antifade montaj reaktif doku korur. NOT: gün içinde hızlı bir şekilde arka plan flüoresan artar; en geç 2 gün içerisinde montaj ortamda gömme sonra mikroskobik analiz nedenle tavsiye edilir. Özel bakım Doğru odak düzlemi ararken ciddi hazırlık zarar verebilir, çünkü doku sıkmak için değil alınmalıdır. Nedeniyle out-of-odak floresan, konfokal mikroskobu ile daha fazla incelenmesi tavsiye edilir. </li></ol>

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The authors have nothing to disclose.

The en face preparation technique described in this protocol provides a powerful tool for the detailed analysis of the olfactory system. So far, most studies characterizing the localization of signaling proteins use immunostainings of cryosections. They present a good overview of the olfactory epithelium, and protein expression in distinct cell types or regions can be easily identified. However, expression in olfactory cilia is sometimes hard to detect. Even if ciliary localization is obvious, cryosections offer...

burun boşluğunda koku epiteli fare 6-10.000.000 bipolar koku duyu nöronları 1 içerir. Her koku nöron ifade 1.200 koku reseptör gen birini seçer. Koku tespiti daha sonra 4 olf koku spesifik G protein Gα üzerinden adenilat siklaz tip III (ACIII) 3 aktive bir koku reseptörü 2, bağlanma odorant başlar. siklik adenozin monofosfat (cAMP) &#39;de elde edilen artış açan bir nükleotid kapılı seçici olmayan katyon kanalı Ca2 + ve Na +, ve daha sonra, Ca girişine yol, (CNG) 2 + akışı, bir Ca + 2 aktif Cl açılmasına neden olan siklik - Kanal 5,6. dışarı doğru Cı Elde - akışı, yüksek hücre içi Cl kolaylaştırılır -, Na + / K + / Cl ile olası emme -, - ortak taşıyıcı NKCC1, konsantrasyon sabit Cl muhafazaCI - / HCO3 - değiştirici SLC4A1 ve belki ek henüz tespit edilmesi taşıyıcıları 6-8. Bipolar koku nöronlar tek dalsız koku ampul doğrudan proje akson ve epitel yüzeyine uzanan ve özel bölmesi, dendritik topuzu gibi biten bir dendrit vardır. Kadar um 50-60 kadar bir uzunluğa ulaşabilir, bu topuz, 10-30 kirpikler itibaren, epitel yüzeyi 9 kaplayan mukus içine sızmak. Kanonik sinyal iletimi akışının Proteinler ağırlıklı bu kirpikler zarında lokalize edilmektedir. epitel artan duyusal yüzey Koku moleküllerini tespit etme yeteneğine güçlendirir. Nedeniyle duyusal nöronların yoğunluğunun, komşu dendritik kolları uzanan kirpikler içe. Epitelyumunun yüzeyi üzerine, koku reseptörlerinin farklı ifade eden farklı nöron gelen kirpikler rastgele karışımı içinde bu karışmasının sonucu. algılama ve hücreduyusal nöronların bir alt kümesi, sadece mevcut siliyer proteinlerin nitlerin ayırma cryosections de zordur. Halinde kriyoseksiyonlar kirpikler ortalama uzunluğundan daha tipik olarak daha ince olduğu için ek olarak, kirpikler boyunca bu proteinlerin kesin yeri, hemen hemen mümkün değildir. Koku nöronlarda kadar uncharacterized membran proteinlerinin siliyer lokalizasyonu soruşturma etkinleştirmek için, biz kirpikler protein lokalizasyonu detaylı analizini sağlayan bir tr yüz hazırlama tekniklerini optimize. Kısaca, fare kurban ve baş orta hatta yakın bölünür. Kıkırdakçıklar burun ve frontal kemik septum maruz bırakmak için çıkarılır. astar epitel koku kısmı ile septum burun boşluğuna tüm bağlantıları keserek gevşetilir. Ringer çözeltisi ile doldurulmuş bir petri kabına septum konulduktan sonra, epitel kaplanmış bir cam slayt aktarılır und soyulur. Kısa bir fixat ardındantaşıma kırılgan doku hasarı önlemek için mümkün olduğunca nazik ise iyon adım, immün işlemleri yapılabilir. Biz klasik cryosections ve tarif tr yüz hazırlık koku kirpikler iki farklı membran proteinlerinin boyama karşılaştırarak elde çözünürlük göstermektedir.

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			<td style="width:167px;">sharp and blunt end, multiple suppliers</td>
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			<td height="21" style="height:21px;width:216px;">Polysine coated slides</td>
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			<td height="42" style="height:42px;width:216px;">primary antibody Goat anti-Kirrel2</td>
			<td style="width:127px;">R&#38;D Systems</td>
			<td style="width:119px;">AF2930</td>
			<td style="width:167px;">1:200</td>
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			<td height="42" style="height:42px;width:216px;">primary antibody Rabbit anti-mOR-EG</td>
			<td style="width:127px;">Baumgart et al., 2014</td>
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			<td style="width:167px;">1:200</td>
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			<td height="21" style="height:21px;width:216px;">secondary antibodies</td>
			<td style="width:127px;">Life Technologies</td>
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			<td height="42" style="height:42px;width:216px;">Mounting medium, ProLong Gold antifade reagent</td>
			<td style="width:127px;">Life Technologies</td>
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			<td height="42" style="height:42px;width:216px;">Paraformaldehyde</td>
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			<td align="right" style="width:119px;">441244</td>
			<td style="width:167px;">toxic, work under fume hood</td>
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whole mount labeling of cilia in the main olfactory system of mice

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single dendrite to the surface of the epithelium, ending in a structure called dendritic knob. Cilia emanate from this knob into the mucus covering the epithelial surface. The proteins of the olfactory signal transduction cascade are mainly localized in the ciliary membrane, being in direct contact with volatile substances in the environment. For a detailed understanding of olfactory signal transduction, one important aspect is the exact morphological analysis of signaling protein distribution. Using light microscopical approaches in conventional cryosections, protein localization in olfactory cilia is difficult to determine due to the density of ciliary structures. To overcome this problem, we optimized an approach for whole mount labeling of cilia, leading to improved visualization of their morphology and the distribution of signaling proteins. We demonstrate the power of this approach by comparing whole mount and conventional cryosection labeling of Kirrel2. This axon-guidance adhesion molecule is known to localize in a subset of sensory neurons and their axons in an activity-dependent manner. Whole mount cilia labeling revealed an additional and novel picture of the localization of this protein.

Yüz hazırlıkları tr Koku epitel olan yerelleştirme cryosections analizinden sonra belirsizdir proteinlerin detaylı soruşturma izin, duyusal nöronların kirpikler proteinlerin yerelleştirme incelemek için kullanılabilir. Bu sorun, Irre benzeri protein 2 (Kirrel2) olarak Kin için boyama durumunda örnek olarak gösterilebilir. (Aynı zamanda Neph3 olarak da adlandırılır) Kirrel2 bir homofilik yapışma proteini olarak zar proteinleri ve fonksiyonların immünoglobulin (Ig) süper ailesinin...

Watch this Scientific Journal Video about Whole Mount Labeling of Cilia in the Main Olfactory System of Mice at JoVE.com

Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Fareler Ana Koku Sistemde Cilia Whole Dağı Etiketleme

The mouse olfactory system comprises 6-10 million olfactory sensory neurons in the epithelium lining the nasal cavity. Olfactory neurons extend a single ...

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Research

JoVE Journal

Neuroscience

11.0K Görüntüleme.  Universitaetsklinikum Jena. Cilia of olfactory sensory neurons contain proteins of the signal transduction cascade, but a detailed spatial analysis of their distribution is difficult in cryosections. We describe here an optimized approach for whole mount labeling and en face visualization of ciliary proteins.

Video: Whole Mount Labeling of Cilia in the Main Olfactory System of Mice

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the procedure for the visualization of Drosophila embryonic nervous system using antibodies to neuronal proteins. Since the entire embryonic peripheral nervous and central nervous systems are well characterized at the level of individual cells (Dambly-Chaudi&egrave;re et al., 1986; Bodmer et al., 1987; Bodmer et al., 1989), any aberrations to these systems can be easily identified using antibodies to different neuronal proteins. The developing embryos are collected at certain times to ensure that the embryos are in the proper developmental stages for visualization. After collection, the outer layers of the embryo, the chorion membrane and the vitelline envelope that surrounds the embryo, are removed before fixation. Embryos are then incubated with neuronal antibodies and visualized using fluorescently labeled secondary antibodies. Embryos at stages 12-17 are visualized to access the embryonic nervous system. At stage 12 the CNS germ band starts shortening and by stage 15 the definitive pattern of the commissure has been achieved. By stage 17 the CNS contracts and the PNS is fully developed (Campos-Ortega et al. 1985). Thus changes in the pattern of the PNS and CNS can be easily observed during these developmental stages.

Visualization of the Embryonic Nervous System in Whole-mount Drosophila Embryos

We describe the procedure to prepare staged Drosophila embryos for the visualization of the embryonic nervous system during embryogenesis.

Tüm montaj Embriyonik Sinir Sistemi Görselleştirme Drosophila Embriyolar

The Drosophila embryo is an attractive model system for investigating the cellular and molecular basis of neuronal development. Here we describe the ...

Nörobilim

Flash Photolysis of Caged Compounds in the Cilia of Olfactory Sensory  Neurons

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. Caged compounds are molecules made physiologically inactive by a chemical cage that can be broken by a flash of ultraviolet light. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged compounds for the study of olfactory transduction in dissociated mouse olfactory sensory neurons. The process of olfactory transduction (Figure 1) takes place in the cilia of olfactory sensory neurons, where odorant binding to receptors leads to the increase of cAMP that opens cyclic nucleotide-gated (CNG) channels2. Ca entry through CNG channels activates Ca-activated Cl channels. We show how to dissociate neurons from the mouse olfactory epithelium3 and how to activate CNG channels or Ca-activated Cl channels by photolysis of caged cAMP4 or caged Ca5. We use a flash lamp6,7 to apply ultraviolet flashes to the ciliary region to uncage cAMP or Ca while patch-clamp recordings are taken to measure the current in the whole-cell voltage-clamp configuration8-11.

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds. Here, we show how to obtain patch-clamp recordings combined with photolysis of caged cAMP or caged Ca for the study of olfactory transduction in dissociated mouse olfactory sensory neurons.

Koku Duyu Nöronlar Cilia Kafesli Bileşiklerin Flash Fotoliz

Photolysis of caged compounds allows the production of rapid and localized increases in the concentration of various physiologically active compounds1. ...

DiI-Labeling of DRG Neurons to Study Axonal Branching in a Whole Mount Preparation of Mouse Embryonic Spinal Cord

Here we present a technique to label the trajectories of small groups of DRG neurons into the embryonic spinal cord by diffusive staining using the lipophilic tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)1. The comparison of axonal pathways of wild-type with those of mouse lines in which genes are mutated allows testing for a functional role of candidate proteins in the control of axonal branching which is an essential mechanism in the wiring of the nervous system. Axonal branching enables an individual neuron to connect with multiple targets, thereby providing the physical basis for the parallel processing of information. Ramifications at intermediate target regions of axonal growth may be distinguished from terminal arborization. Furthermore, different modes of axonal branch formation may be classified depending on whether branching results from the activities of the growth cone (splitting or delayed branching) or from the budding of collaterals from the axon shaft in a process called interstitial branching2 (Fig. 1).
The central projections of neurons from the DRG offer a useful experimental system to study both types of axonal branching: when their afferent axons reach the dorsal root entry zone (DREZ) of the spinal cord between embryonic days 10 to 13 (E10 - E13) they display a stereotyped pattern of T- or Y-shaped bifurcation. The two resulting daughter axons then proceed in rostral or caudal directions, respectively, at the dorsolateral margin of the cord and only after a waiting period collaterals sprout from these stem axons to penetrate the gray matter (interstitial branching) and project to relay neurons in specific laminae of the spinal cord where they further arborize (terminal branching)3. DiI tracings have revealed growth cones at the dorsal root entry zone of the spinal cord that appeared to be in the process of splitting suggesting that bifurcation is caused by splitting of the growth cone itself4 (Fig. 2), however, other options have been discussed as well5.
This video demonstrates first how to dissect the spinal cord of E12.5 mice leaving the DRG attached. Following fixation of the specimen tiny amounts of DiI are applied to DRG using glass needles pulled from capillary tubes. After an incubation step, the labeled spinal cord is mounted as an inverted open-book preparation to analyze individual axons using fluorescence microscopy.

The stereotyped projections of sensory afferents into the rodent spinal cord offer an easily accessible experimental system to study axonal branching through the tracing of single axons.

Fare Embriyonik Omurilik Tüm Dağı Hazırlık aksonal dallanma Eğitim DRG Nöronlar DII-Etiketleme

Here we  present a technique to label the trajectories of small groups of DRG neurons  into the embryonic spinal cord by diffusive staining using the ...

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

In this article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the lipophilic fluorescent membrane marker DiI. This method allows the visualization of neuronal cell morphology in great detail. It is possible to label any cell in the CNS: cell bodies of target neurons are visualized under DIC optics or by expression of a fluorescent genetic marker such as GFP. After labeling, the DiI can be transformed into a permanent brown stain by photoconversion to allow visualization of cell morphology with transmitted light and DIC optics. Alternatively, the DiI-labeled cells can be observed directly with confocal microscopy, enabling genetically introduced fluorescent reporter proteins to be colocalised. The technique can be used in any animal, irrespective of genotype, making it possible to analyze mutant phenotypes at single cell resolution.

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

We present a technique for labeling single neurons in the central nervous system (CNS) of Drosophila embryos, which allows the analysis of neuronal morphology by either transmitted light or confocal microscopy.

Merkez Sinir Sistemi Tek Hücreler Etiketleme<em&gt; Drosophila melanogaster</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

Angiogenesis is the complex process of new blood vessel formation defined by the sprouting of new blood vessels from a pre-existing vessel network. Angiogenesis plays a key role not only in normal development of organs and tissues, but also in many diseases in which blood vessel formation is dysregulated, such as cancer, blindness and ischemic diseases. In adult life, blood vessels are generally quiescent so angiogenesis is an important target for novel drug development to try and regulate new vessel formation specifically in disease. In order to better understand angiogenesis and to develop appropriate strategies to regulate it, models are required that accurately reflect the different biological steps that are involved. The mouse neonatal retina provides an excellent model of angiogenesis because arteries, veins and capillaries develop to form a vascular plexus during the first week after birth. This model also has the advantage of having a two-dimensional (2D) structure making analysis straightforward compared with the complex 3D anatomy of other vascular networks. By analyzing the retinal vascular plexus at different times after birth, it is possible to observe the various stages of angiogenesis under the microscope. This article demonstrates a straightforward procedure for analyzing the vasculature of a mouse retina using fluorescent staining with isolectin and vascular specific antibodies.

Whole Mount Immunofluorescent Staining of the Neonatal Mouse Retina to Investigate Angiogenesis In vivo

The neonatal murine retina provides a well characterized physiological model of angiogenesis, which permits investigations of the roles of different genes or drugs that modulate angiogenesis in an in vivo context. Immunofluorescent staining to accurately visualize the vascular plexus is pivotal to the success of these types of studies.

Anjiyogenez Araştırma Yenidoğan Fare Retina Tüm Dağı Immunofluorescent Boyama<em&gt; In vivo</em

Angiogenesis  is the complex process of new blood vessel formation defined by the sprouting  of new blood vessels from a pre-existing vessel network. ...

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat muscles can offer significant advantage over traditionally used thicker muscles, such as those from the hind limb (e.g. gastrocnemius). Thin muscles allow for comprehensive overview of the entire innervation pattern for a given muscle, which in turn permits identification of selectively vulnerable pools of motor neurons. These muscles also allow analysis of parameters such as motor unit size, axonal branching, and terminal/nodal sprouting. A common obstacle in using such muscles is gaining the technical expertise to dissect them. In this video, we detail the protocol for dissecting the transversus abdominis (TVA) muscle from young mice and performing immunofluorescence to visualize axons and neuromuscular junctions (NMJs). We demonstrate that this technique gives a complete overview of the innervation pattern of the TVA muscle&#160;and can be used to investigate NMJ pathology in a mouse model of the childhood motor neuron disease, spinal muscular atrophy.

Dissection of the Transversus Abdominis Muscle for Whole-mount Neuromuscular Junction Analysis

In this video we demonstrate a protocol for dissection of the transversus abdominis muscle of the mouse and use immunofluorescence and microscopy to visualize neuromuscular junctions.

Tüm Montajlı Nöromüsküler Kavşak Analizi için Enine Abdominis Kasının Diseksiyonu

Analysis of neuromuscular junction morphology can give important insight into the physiological status of a given motor neuron. Analysis of thin flat ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

In Koku Reseptör Nöronlar Tüm Dağı ile immün<em&gt; Drosophila</em&gt; Anten

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Live Imaging of the Ependymal Cilia in the Lateral Ventricles of the Mouse Brain

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various neurological deficits. The current ex vivo live imaging of motile ependymal cilia technique allows for a detailed study of ciliary dynamics following several steps. These steps include: mice euthanasia with carbon dioxide according to protocols of The University of Toledo&#8217;s Institutional Animal Care and Use Committee (IACUC); craniectomy followed by brain removal and sagittal brain dissection with a vibratome or sharp blade to obtain very thin sections through the brain lateral ventricles, where the ependymal cilia can be visualized. Incubation of the brain&#8217;s slices in a customized glass-bottom plate containing Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)/High-Glucose at 37 &#176;C in the presence of 95%/5% O2/CO2 mixture is essential to keep the tissue alive during the experiment. A video of the cilia beating is then recorded using a high-resolution differential interference contrast microscope. The video is then analyzed frame by frame to calculate the ciliary beating frequency. This allows distinct classification of the ependymal cells into three categories or types based on their ciliary beating frequency and angle. Furthermore, this technique allows the use of high-speed fluorescence imaging analysis to characterize the unique intracellular calcium oscillation properties of ependymal cells as well as the effect of pharmacological agents on the calcium oscillations and the ciliary beating frequency. In addition, this technique is suitable for immunofluorescence imaging for ciliary structure and ciliary protein localization studies. This is particularly important in disease diagnosis and phenotype studies. The main limitation of the technique is attributed to the decrease in live motile cilia movement as the brain tissue starts to die.

Using high-resolution differential interference contrast (DIC) microscopy, an ex vivo observation of the beating of motile ependymal cilia located within the mouse brain ventricles is demonstrated by live-imaging. The technique allows a recording of the unique ciliary beating frequency and beating angle as well as their intracellular calcium oscillation pacing properties.

Fare Beyin lateral ventrikül Ependimal Cilia Canlı Görüntüleme

Multiciliated ependymal cells line the ventricles in the adult brain. Abnormal function or structure of ependymal cilia is associated with various ...

Whole Mount Dissection and Immunofluorescence of the Adult Mouse Cochlea

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a spiral shape and have a tonotopic gradient for sound detection. The mouse cochlea is approximately 6 mm long and often divided into three turns (apex, middle, and base) for analysis. To investigate cell loss, cell division, or mosaic gene expression, the whole mount or surface preparation of the cochlea is useful. This dissection method allows visualization of all cells within the organ of Corti when combined with immunostaining and confocal microscopy to image cells at different planes in the z-axis. Multiple optical cross-sections can also be obtained from these z-stack images. In addition, the whole mount dissection method can be used for scanning electron microscopy, although a different fixation method is needed. Here, we present a method to isolate the organ of Corti as three intact cochlear turns (apex, middle, and base). This method can be used for mice ranging from one week of age through adulthood and differs from the technique used for neonatal samples where calcification of the cochlea is incomplete. A slightly modified version can be used for dissection of the rat cochlea. We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies.

We present a method to isolate the adult organ of Corti as three intact cochlear turns (apex, middle, and base). We also demonstrate a procedure for immunostaining with fluorescently tagged antibodies. Together these techniques allow the study of hair cells, supporting cells, and other cell types found in the cochlea.

Tüm Dağı Diseksiyon ve Yetişkin Fare Kohlea immünofloresan

The organ of Corti, housed in the cochlea of the inner ear, contains mechanosensory hair cells and surrounding supporting cells which are organized in a ...

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

This article describes how to inject viral vectors into the mouse frontal cortex to test behavioral assays that require GPCR heteromeric formation.

Kimerik Yapılar HSV-aracılı Transgen İfade Farelerde GPCR Heteromers Davranışsal Fonksiyon Eğitim için

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...