Yazarlar anlayışlı tartışma ve önerileriniz için TrkA taulacZ fareler Dr. Annette Markus Dr. Louis Reichardt teşekkür etmek istiyorum. Bu çalışma Burke Vakfı yanı sıra Whitehall Vakfı araştırma bursu 2010-08-61, Yaşam Vakfı (WFL-ABD-028/14) için Wings bir araştırma hibe başlangıç ​​fonları tarafından desteklenen, gelen ZB1-1102-1 hibe Christopher &amp; Dana Reeve Vakfı, ve JZ Ulusal Göz Enstitüsü hibe 1R01EY022409 ve 3R01EY022409-01S1. KJO bir Goldsmith üyesidir.

NOT: Tüm işlemler Laboratuvar Hayvanları Kullanımı ve Bakımı NIH Kılavuzu ile uyumlu. Hayvan protokolü Weill Cornell Medical College IACUC tarafından onaylanmıştır. 1. Doku Hazırlanması <ol><li> Servikal dislokasyon 15 ile zamanlanmış-gebelik kadın Euthanize. Ayrı ayrı soğutulmuş fosfat tamponlu tuz (PBS) ile doldurulmuş bir 6-yuvalı plakasının gözenekleri içinde zamanlanmış hamilelik kadın ve yer embriyolardan E18 embriyoları - embriyonik E16 ayır. </li><li> Soğuk PBS embriyolar durulayın. </li><li> Daha sonra genotipleme için önceden hazırlanmış bir proteinaz K solüsyonunda embriyo ve yer amniyotik zarın küçük bir kısmını çıkarmak. Amniyotik zar kaybolur Alternatif olarak, eğer embriyo kuyruk ucu küçük bir bölümünü kaldırmak ve proteinaz K çözeltisi yerleştirin </li><li> Tüm böcek işaretçilerine (yan başına en az 100) kullanılarak embriyo çevresindeki deri içine delikleri Poke. </li><li> (PFA) taze% 2 paraformaldehid ekleyin yavaş yavaş PBS çıkarın vekuyu pH 7.4. </li><li> Yavaşça 4 ° C&#39;de 2 saat boyunca çalkalanır. </li><li> PBS içinde iki kere embriyo yıkayın ve boyama kadar 4 ° C de PBS içinde depolar. </li></ol> 2. Genotipleme <ol><li> Üreticinin talimatlarına göre proteinaz K karışımı içinde amniyotik membranlar veya embriyo kuyrukları daldırın. </li><li> 10 dakika boyunca 56 ° C&#39;de inkübe edilir. </li><li> Bir ticari olarak temin edilebilir DNA saflaştırma kiti kullanılarak DNA ekstrakte edin. </li><li> Gen spesifik sens ve antisens primerler (10 uM), her biri 0.5 ul, 2 ul dNTP karışımı ile birleştirmek, genomik DNA solüsyonu arıtıldı toplam 200 ul, 0.5 ul alın (dATP 2.5 mM, dCTP, dGTP ve dTTP) 0.2 ul Taq polimeraz (5 U / ul) ve 2 ul PCR tamponu (10x), 20 ul toplam hacim H2O ekleyin. NOT: Primer dizileri aşağıda (Tablo 1): TrkA: TrkA_F: GCG GGC GCC KİK JGK ATG, TrkA_R: GAA GCC KİK TGC GCG GTT CTG CCA GGG TG; PCR fragment uzunluğu: 400 baz çifti (bp). Bax: In5R: GTT GAC CAG AGT GGC GTA GG, Ex5F: TGA TCA GAA CCA TCA TG, neoR: CCG CTT CCA TTG CTC AGC GG; PCR fragmanı uzunlukları: vahşi tip, 304 bp; Bax boş, 507 bp. TauLacZ: lacZup: ACA ACG TCG TGA GTG GGA AAA, lacZdown: ATC AAC ATT AAA TGT GAG CGA G; PCR parçası uzunluğu: 360 bp. </li><li> TrkA aşağıdaki program ile bir PCR çalıştırın: Aşama 1: 1 dakika 95 ° C, aşama 2 için: 30 saniye için 72 ° C: 68 ° C, aşama 4&#39;te, 20 saniye: 95 ° C, aşama 3 10 san. 40 döngü için 4 - Tekrar 2 adımları. <ol><li> LacZ ve Bax, aşağıdaki program ile bir PCR çalıştırın: Aşama 1: 1 dakika 95 ° C de, aşama 2: 10 saniye 95 ° C, aşama 3: 1 dak 54 ° C, aşama 4: 72 ° C&#39;da 1 dakika 35 döngü için 4 - ° C, tekrar adımları 2. </li></ol></li><li> DNA merdiveni bir şerit 20 dakika süre ile 1x Tris-Asetat-EDTA tampon maddesi içinde 100 V de,% 2 agaroz jel üzerinde yapılmıştır, PCR numuneleri fragmanı uzunlukları değerlendirmek. </li><li> Vahşi tip TrkA, T varlığı için embriyolar analizRKA-tauLacZ ve Bax boş allel. Bax - / -, deneysel sorular bağlı: - - / ve TrkA taulacZ / taulacZ Bax: Deneysel embriyoların genotipleri TrkA taulacZ / WT vardır. TauLacZ muhabiri eksik Embriyolar aksonal boyama için olumsuz olacak ve bu nedenle boyama işlemini kalibre etmek için negatif kontroller hizmet edebilir. </li></ol> 3. Embriyo Boyama <ol><li> 3.8 - 3.2 bölümlerde de aşağıda tarif edildiği gibi, bazı modifikasyonlarla, lacZ boyama için bir ticari kit kullanın. Burada, Millipore kiti kullanın. </li><li> Oda sıcaklığında hafifçe çalkalanarak, en az 1 saat süre ile tampon çözeltisi içinde, embriyolar bırakın. </li><li> Doğrudan, oda sıcaklığında hafifçe çalkalanarak, en azından 15 dakika için, Tampon B embriyolar bırakın. </li><li> Embriyolar Tampon A / B, 37 ° C&#39;de önceden sıcak Doku baz çözelti içinde iken. Her embriyo için doku baz çözeltisi 5 ml kullanın. </li><li> Embriyolar Tampon A / B olarak da, yeni 5-br hazırlanmasıomo-% 100 dimetil sülfoksit (DMSO) içinde 40 mg / ml arasında bir konsantrasyonda 3-indolil α-D-galaktopiranosid (X-gal) 4-klorofenil. </li><li> 1 mg / ml bir konsantrasyonda önceden ısıtılmış Doku baz çözeltisi içine X-gal ile seyreltilir ve bir cam sintilasyon şişesine X-gal / Doku baz karışımı 5 ml ilave ediniz. </li><li> X-gal / Doku baz karışımını ihtiva eden bir cam sintilasyon şişesine embriyolar aktarın. Parafilm ile kapağı kapatın ve boyası olup olmadığını kontrol, bir gece boyunca 37 ° C&#39;de inkübe edin. </li><li> Taze% 4 PFA, pH 7.4 ile Postfix&#39;i embriyolar. Yavaşça 4 ° C de 4 saat süre ile çalkalanır. </li><li> Soğuk PBS iki kez embriyolar durulayın. </li></ol> 4. Doku Dehidrasyon ve Takas <ol><li> Cam şişeler içinde çalkalama ile oda sıcaklığında 30 dakika boyunca% 50 metanol /% 50 PBS karışımı içinde yer embriyolar. </li><li> Cam şişeler içinde çalkalanarak, oda sıcaklığında 30 dakika boyunca% 75 metanol /% 25 PBS kurutmak için devam edin. </li><li> % 90 kurutmak devam metanol /% 10 pCam şişeler içinde çalkalanarak, oda sıcaklığında, 30 dakika, BS. </li><li> Cam şişeler içinde çalkalanarak, oda sıcaklığında 30 dakika boyunca (2x) için% 100 metanol içinde kurutmak için devam edin. </li><li> 1 (v: v) benzoil alkol ve benzoil benzoat karışımı embriyolar dehidrasyon iken, bir 1 hazırlayın. Not: Benzoil alkol ve benzoil benzoat toksiktir. </li><li> 1: 1 embriyolar daldırın (v: v),% 100 metanol karışımı: 15 dakika boyunca% 100 BABB. BABB plastik eriyecektir çünkü sadece camını kullanın. </li><li> Tamamen açık doku% 100 Babb embriyolar daldırın ve temizlenmiş embriyo X-gal boyama görselleştirmek için. NOT: kısa sürede X-gal leke zamanla solmaya çünkü görüntü. </li></ol> 5. Tüm Dağı Görüntüleme <ol><li> Fotoğrafçılık ile metal forseps için uygun açılarda,% 100 Babb dalmış embriyo, stabilize. Işık-up ve altında ışık ışık kaynaklarının bir arada kullanarak Aydınlatmak. </li><li> Diseksiyon mikroskobu görüntüleri EdinmeBir dijital kamera ile donatılmış. 0.5 mm aralıklı Z ekseni boyunca beş ardışık odak düzlemleri birden fazla parlak bir alan poz alın. Pozlama süreleri ve f-durur aydınlatma ve kamera özelliklerine göre değişir ve her kurulum için optimize edilmesi gerekebilir. </li><li> Bindirme görüntüleri işlemek ve fotomontajların oluşturmak için standart görüntü işleme yazılımı kullanın. </li></ol>

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The authors declare that there are no competing interests.

Embriyonik TrkA taulacZ farelerin yukarıda tarif edilen X-gal lekeleme prosedürü sağlam, sabit embriyo uzun mesafe akson çıkıntıların hızlı ve ayrıntılı olarak gösterebilir. Bu fareler akson büyümesi ve nöronal hayatta hem de katkıda bulunabilir mekanizmalar sinyal tarama izin Bax boş arka plan Çünkü. Ilgi transgenik veya nakavt fareler ile çiftleşme aksonal fenotip kapsamlı değerlendirilmesi için izin verir ve daha detaylı bir şekilde akson büyüme sinyalini i...

Kesin nöronal ağların kurulması sinir sisteminin işlevselliği için gerekli karmaşık bir gelişimsel süreçtir. Bu süreçte sorunlar olduğu insan nörolojik hastalıklar 1-3 implike edilmiştir nöronal fonksiyon bozukluğuna yol açar. Memelilerde akson büyümesi ve hedef innervasyon yatan moleküler mekanizmaları incelemek için, iki genetiği değiştirilmiş fare hatları bir arada kullanarak duyusal nöronlar TrkA-ifade aksonal yörüngeleri görselleştirmek için bir protokol geliştirmiştir. TrkA sinir büyüme faktörü NGF için bir alıcı, ve nosiseptif duyu nöronlarının 4 fonksiyonel bir işaretleyici olarak kullanılabilir. TrkA derece erken gelişimi sırasında nosiseptif nöronların ifade ve NGF-bağımlı nöron hayatta kalma, akson büyümesini, arborization ve hedef innervasyon 5-9 aracılık eder. TrkA taulacZ farelerde, vahşi tip TrkA geninin taulacZ sentezleme c değiştirilirfarazi TrkA pozitif nöronlarının akson morfolojisi 11 boyama β-gal (X-gal) ile görüntülenebilir şekilde assette 10. Heterozigot TrkA taulacZ / WT hattını kullanarak, düzenlemek veya in vivo duyusal afferent projeksiyonlar gelişmesine engel olabilir faktörleri inceleyebilirsiniz. Ayrıca, TrkA ifadeleri, bu nedenle, NGF / TrkA sinyal olmadığında mekanizmaları teşvik akson büyümesini belirlemek üzere kullanılabilir homozigot TrkA taulacZ / taulacZ farelerde, yoktur. Nosiseptif nöronlar, NGF / TrkA sadece akson büyümesi için sinyal bağlı olduklarından, aynı zamanda hayatta kalmak için, başka türlü hücre ölümden kurtarmak, embriyonik DRG nöronlarının apoptosisi engellemek için, pro-apoptotik Bax geni eksik bir fare hattı kullanır TrkA sinyallemesinin yokluğunda gözlemlenen. Bax - / - Arka plan 12 Böylece molecu sağlarözellikle akson büyüme 7-9,13-15 etkileyen sinyal yollarının lar diseksiyonu. TrkA yılında - / -: Bax - / - fareler, DRG nöronlar hayatta, ama deride duyu afferent innervasyon tamamen 14,15 kaldırılmıştır. Biz seçici akson projeksiyonlar gelişmesine kendi katkılarını belirlemek için sinyal yolları aktive edebilirsiniz. Bu yöntemin yarar farklı genetik değişiklikler TrkA taulacZ / taulacZ üzerine yetiştirilen zaman aksonal büyüme fenotipleri değişikliklerin değerlendirilmesine olanak sağlamasıdır: Bax - / - veya TrkA taulacZ / WT: Bax - / - kökenden.

<table border="0" cellpadding="0" cellspacing="0" width="465">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:199px;"></td>
			<td style="width:127px;">Company</td>
			<td style="width:139px;">Catalog Number</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PFA</td>
			<td>Sigma-Aldrich</td>
			<td>P6418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life Tech</td>
			<td>10010-023</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution A</td>
			<td>Millipore</td>
			<td>BG-6-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Rinse Solution B</td>
			<td>Millipore</td>
			<td>BG-7-B</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue Stain Base Solution</td>
			<td>Millipore</td>
			<td>BG-8-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>B4252</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintiallation vial</td>
			<td>Kimble Chase</td>
			<td>74500-20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Incubator</td>
			<td>Labline</td>
			<td>Model 120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insect pins</td>
			<td>FST</td>
			<td>26000-30</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well dish</td>
			<td>USA Scientific</td>
			<td>CC7672-7506</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primers</td>
			<td>IDT</td>
			<td>custom DNA primers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara dNTP mixture</td>
			<td>Takara</td>
			<td>4030</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Takara LA buffer</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Takara LA Taq</td>
			<td>Takara</td>
			<td>RR002A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR machine</td>
			<td>Bio-Rad&#160;</td>
			<td>DNA Engine Dyad</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl alcohol</td>
			<td>Sigma-Aldrich</td>
			<td>B-1042</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Benzyl benzoate</td>
			<td>Sigma-Aldrich</td>
			<td>B-6630</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting microscope</td>
			<td>Leica</td>
			<td>M205A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Camera</td>
			<td>Leica</td>
			<td>DFC310FX</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ring light&#160;</td>
			<td>Leica&#160;</td>
			<td>MEB110</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Photoshop</td>
			<td>Adobe</td>
			<td>Photoshop 4.0</td>
		</tr>
	</tbody>
</table>

whole-mount imaging of mouse embryo sensory axon projections

embriyolarda tam uzunlukta nöronal projeksiyonlar görselleştirme nöronal ağlar nasıl geliştiğini memeli bir anlayış kazanmak esastır. Burada yerinde birkaç genetik manipüle fare hatları kullanarak fenotipik özelliklerini değerlendirmek için dorsal kök ganglion (DRG) akson projeksiyonlar bir alt kümesini etiketlemek için bir yöntem açıklanmaktadır. TrkA pozitif nöronların ağrı sinyallerinin iletimi için özel nosiseptör nöronlar vardır. Biz sağlam fare embriyo tüm TrkA-pozitif çevresel akson yörüngeleri etiketlemek için bir TrkA taulacZ fare hattını kullanmak. Biz daha bağımsız nöronal sağkalım üzerine genetik manipülasyonlar olası etkilerinin büyüme ile ilgili soruları değerlendirmek amacıyla, esasen nöronal apoptoz ortadan kaldıran bir Bax boş bir arka plan üzerine TrkA taulacZ hattını doğurmak. Daha sonra, ilgi genetiği değiştirilmiş fareler TrkA Taul ile yetiştirilenACZ / Bax boş hattı ve daha sonra burada tarif edilen teknikler kullanılarak çalışma için hazırdır. Bu sunum, tüm montaj hazırlanmasında tam uzunlukta aksonal yörüngeleri görünüm için izin diseksiyon, doku hazırlanması, boyama ve temizleme sırasında detaylı fare üreme planları hakkında bilgi, genotiplendirmesi içerir.

TrkA ağ / taulacZ genotipleri: Bax - / - ve TrkA taulacZ / taulacZ: Bax - / - embriyolar açıkça standart PCR genotipleme ile tespit edilebilir (Şekil 1). X-gal lekeleme görüntüler geleneksel lekeli embriyolar (Şekiller 2, 3a) &#39;da deri altından periferik aksonal milleri ayrıntılı ve doku temizleme sonra embriyo boyunca (Şekil 3b, 4). - / - Hattı nörona özgü Nestin pr...

Watch this Scientific Journal Video about Whole-mount Imaging of Mouse Embryo Sensory Axon Projections at JoVE.com

Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Fare Embriyo Duyu Akson Projeksiyonlar tüm montaj Görüntüleme

The visualization of full-length neuronal projections in embryos is essential to gain an understanding of how mammalian neuronal networks develop. Here we ...

whole-mount-imaging-of-mouse-embryo-sensory-axon-projections

Research

JoVE Journal

Neuroscience

10.0K Görüntüleme.  Weill Medical College of Cornell University. We present here an optimized protocol to genotype, stain and prepare fetal mice for the imaging of peripheral nociceptor axon projections in the whole animal, as an effective method to assess sensory axon growth phenotypes in developing genetically engineered mice.

Video: Whole-mount Imaging of Mouse Embryo Sensory Axon Projections

Reproducible Mouse Sciatic Nerve Crush and Subsequent Assessment of Regeneration by Whole Mount Muscle Analysis

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative response mounted by PNS neurons thereby possibly illuminating the failures of CNS regeneration1. Sciatic nerve crush (axonotmesis) is one of the most common models of peripheral nerve injury in rodents2. Crushing interrupts all axons but Schwann cell basal laminae are preserved so that regeneration is optimal3,4. This allows the investigator to study precisely the ability of a growing axon to interact with both the Schwann cell and basal laminae4. Rats have generally been the preferred animal models for experimental nerve crush. They are widely available and their lesioned sciatic nerve provides a reasonable approximation of human nerve lesions5,4. Though smaller in size than rat nerve, the mouse nerve has many similar qualities. Most importantly though, mouse models are increasingly valuable because of the wide availability of transgenic lines now allows for a detailed dissection of the individual molecules critical for nerve regeneration6, 7. Prior investigators have used multiple methods to produce a nerve crush or injury including simple angled forceps, chilled forceps, hemostatic forceps, vascular clamps, and investigator-designed clamps8,9,10,11,12. Investigators have also used various methods of marking the injury site including suture, carbon particles and fluorescent beads13,14,1. We describe our method to obtain a reproducibly complete sciatic nerve crush with accurate and persistent marking of the crush-site using a fine hemostatic forceps and subsequent carbon crush-site marking. As part of our description of the sciatic nerve crush procedure we have also included a relatively simple method of muscle whole mount we use to subsequently quantify regeneration.

In this report we describe a method to crush mouse sciatic nerve. This method uses readily available hemostatic forceps and easily and reproducibly produces complete sciatic nerve crush. In addition, we describe a method to prepare muscle whole mounts suitable for analysis of nerve regeneration after sciatic nerve crush.

Tüm Dağı Muscle Analizi ile tekrarlanabilir Fare Siyatik Sinir Ezilme ve Rejenerasyon daha sonraki Değerlendirme

Regeneration in the peripheral nervous system (PNS) is widely studied both for its relevance to human disease and to understand the robust regenerative ...

Nörobilim

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 ...

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. ...

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 ...

Flat Mount Imaging of Mouse Skin and Its Application to the Analysis of Hair Follicle Patterning and Sensory Axon Morphology

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel cell clusters), sebaceous glands, nerves and nerve endings, and capillaries. The spatial arrangement of these structures is tightly controlled on a microscopic scale - as seen, for example, in the orderly arrangement of cell types within a single hair follicle - and on a macroscopic scale - as seen by the nearly identical orientations of thousands of hair follicles within a local region of skin. Visualizing these structures without physically sectioning the skin is possible because of the 2-dimensional geometry of this organ. In this protocol, we show that mouse skin can be dissected, fixed, permeabilized, stained, and clarified as an intact two dimensional object, a flat mount. The protocol allows for easy visualization of skin structures in their entirety through the full thickness of large areas of skin by optical sectioning and reconstruction. Images of these structures can also be integrated with information about position and orientation relative to the body axes.

Mammalian skin contains a diverse array of structures - such as hair follicles and nerve endings - that exhibit distinctive patterns of spatial organization. Analyzing skin as a flat mount takes advantage of the 2-dimensional geometry of this tissue to produce full-thickness high-resolution images of skin structures.

Fare Cilt ve Saç Folikül Desenlendirme ve Duyusal Akson Morfoloji Analizi Uygulanması Düz ​​Görüntüleme Dağı

Skin is a highly heterogeneous tissue. Intra-dermal structures include hair follicles, arrector pili muscles, epidermal specializations (such as Merkel ...

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.

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 ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Görüntüleme Ca<sup&gt; 2+</supFare Retina Koni fotoreseptör Akson Terminalleri içinde&gt; Dinamikleri

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

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 ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Optik Koherens tomografi: Fare retina Ganglion hücreleri Vivo içinde görüntüleme

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Quadruple Immunostaining of the Olfactory Bulb for Visualization of Olfactory Sensory Axon Molecular Identity Codes

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory Sensory Neuron (OSN) is a bipolar cell with a single dendrite and a single unbranched axon. An OSN expresses only one Olfactory Receptor (OR) gene, OSNs expressing a given type of OR converge their axons to a few sets of invariant glomeruli in the Olfactory Bulb (OB). A remarkable feature of OSN projection is that the expressed ORs play instructive roles in axonal projection. ORs regulate the expression of multiple axon-sorting molecules and generate the combinatorial molecular code of axon-sorting molecules at the OSN axon termini. Thus, to understand the molecular mechanisms of OR-specific axon guidance mechanisms, it is vital to characterize their expression profiles at the OSN axon termini within the same glomerulus. The aim of this article was to introduce methods for collecting as many glomeruli as possible on a single OB section and for performing immunostaining using multiple antibodies. This would allow the comparison and analysis of the expression patterns of axon-sorting molecules without staining variation between OB sections.

Olfactory sensory neurons express a wide variety of axon-sorting molecules to establish proper neural circuitry. This protocol describes an immunohistochemical staining method to visualize combinatorial expressions of axon-sorting molecules at the axon termini of olfactory sensory neurons.

Koku Meşfatının Olfaktör Duyusal Akson Moleküler Kimlik Kodlarının Görselleştirilmesi İçin Dörtlü İmmün Sulanması

The mouse olfactory system is often used to study mechanisms of neural circuit formation&#12288;because of its simple anatomical structure. An Olfactory ...