Biz minnetle Marder Lab üyeleri kabul etmiş oluyorsunuz. Danışmanı Dr. Havva Marder, Biz ona destek ve cesaret için teşekkür ederim. Bu araştırma, Ulusal Nörolojik Bozukluklar ve İnme Hibeler NS-17.813 ve NS-058.110 Enstitüsü tarafından desteklenmiştir.

1. Brüt Diseksiyon <ol><li> Hayvan donuk duyu ve motor sistemlerinin 30 dakika boyunca buz üzerinde bir yengeç yerleştirin. Buz ile her tarafta onu çevreleyen. Yengeç Kullanım sırasında eldiven giyilmelidir. </li><li> Diseksiyonu pan ve kullanacağınız araçları dışarı atarken brüt diseksiyon için hazırlayın. Rongeurs, kemik kesme makası, küçük makas, dişli forseps, konik kenarı ile spatula, siyah Sylgard kaplı çanak, ve böcek pimleri ihtiyaç vardır. </li><li> Sıkışmasını önlemek ve diseksiyon tavada yerleştirmek için, arka bacak buz kovası yengeç çıkartın. </li><li> Içe her bir büküm ilk pençeleri çıkarın. Sonra aynı büküm hareketi her bacak kaldırmak. </li><li> Rongeurs kullanarak, ağız kapsayan maksilla çıkarın. Ayrıca yumuşak maxillule çıkarın. </li><li> Rongeurs kullanarak tekrar karapaksla lateral posterior sonundan itibaren başlamak ve bunun kenar iyi bir kavrama olsun. Kelepçe rongeurs ile aşağı ve karapaksla bir parça kesiyorum. Spatula açılış girmesi için yeterli karapaksta kaldırmak için emin olun. Gözleri karapaksla kenarından yukarı kaldırarak, diğer tarafta devam edin ve işlemi tekrarlayın. </li><li> Spatula sivri tarafı hafifçe dorsal karapaksla çatı uzak yumuşak iç dokusu kazımak. Spatula STNS zarar görmesini önlemek için karapaksta ile temas halinde tutun. Rongeurs dokusundan ayrılmış karapaksla parçaları kurtulmak için kullanın. Ayrıca bazı yumuşak doku karapaksla doku ekleme adduktor kasları ayrı emin olun ventral karapaksla ayırın. </li><li> Karapaksta çıkıntılı iki küçük kemikçikler vardır kalbinde kadar posterior dorsal karapaksla Break Away. Gibi çok ön mümkün olduğunca kesin. Dorsal karapaksla en ön parça terkedip Cephalon (&quot;yüz&quot;) bir parmak ile sabitleyin. </li><li> Karapaksla ventral tarafta, onlara ekli epistome mandibula çıkarmak için dışa büküm ve dışarı çekerek, mandibula üzerine rongeurs demir bir zamanda çene kemiklerini bir çekin. İki kasları ile uzun kemikçikler, doğru yapılırsa Ayıklanan mandibula eklenmelidir. </li><li> Spatula ile Gözlere yakın Cephalon iki doku ayırın. Doku tabakası tarafından, olası bir katman olarak kemik parçası sırt yakın kesen küçük diseksiyon makas. </li><li> Diseksiyonu pan bir duvara yengeç istirahat ve yerine o güvenliğini (pençeleri için kullanılan olabilir) desteklemek. Herhangi bir doku eklenmiş bırakmamaya dikkat ederek epistome uzak labrum (üst dudak) kesin. Kemik kesme makası kullanarak Cephalon kaldırmak için ventral karapaksla iki diyagonal kesimler yapmak. </li><li> Dişli forseps ile labrum ve ventral tarafta kemik parçası uzak labium (&quot;alt dudak&quot;) kesmek. Sonra yavaşça kalan ventral karapaksla uzak dudak kaldırma. Karapaksla döşeyen doku mide ventral kısmı ayırmak için küçük makas kullanın. Mide yengeç çıkarılan kadar pilorik ampullae görünür olduğunda, bunun her iki tarafta doku uzak mide kesmek. </li><li> Yine de, elin avuç içi karşı mide dinlenme ve ağız içine tuzlu su dökün, forseps ile dudak mide tutarak. Bu mide genişler ve daha kolay açık kesmek için yapar. Aşağı pilorusta ampullae arasındaki kemik parçası ile sağ arasındaki ağız açılması kesin. Sonra iki Kardiyal şubeleri aracılığı ile çapraz aşağı kesti. Mide içinde üç diş ipuçları kesti. </li><li> Mide aşağı mide içinde büyük, siyah, Sylgard kaplı tabağa yerleştirin. Beş köşeli hazırlık böcek iğneli pin ve çanak tüm hazırlık çeker (bkz. Şekil 1), soğuk tuzlu su ile doldurun. </li></ol> 2. İnce Diseksiyon <ol><li> , Mikro-disseksiyon araçları ince diseksiyon için ihtiyaç vardır. Özellikle, forseps ve yaylı makas tungsten iğne ile bir iğne tutucu yanı sıra ihtiyaç vardır. Net, Sylgard kaplı petri ve bazı ince tel pimleri da hazırlanmış olmalıdır. </li><li> En düşük büyütme başlayarak, ağız açıklığı için kullanılan dudak yakın kapakları aşağı iğneleyerek başlar. Hazırlık gergin yapmak ve diğer alanlarda gerekli her türlü pin pimleri geri kalanı yeniden düzenleyin. </li><li> Hazırlık alt kısmını kapsayan benekli hipodermis dikkatlice kaldırın ve iki beyaz nokta ile hipodermis bir yama bırakarak üzerinden bunu düzeltin. </li><li> Hazırlanması alt yakın sarı, bir tutam, membranöz doku kadar soyun. Mvns zayıf bu doku alt bağlıdırlar. O kadar çekilir gibi sarı doku dikkatlice mvns ayırın. Sarı doku takip ve beyin yanında kesmek. </li><li> Beyin çıkmadan kalın süreçlerini takip uzatmak laterally Bunlar commisural sinirler vardır. Commissural sinir sonuna ulaşıldığında, beynin yan uzak kesilmiş, off-beyaz süngerimsi doku kitle çıkarın. </li><li> Iyonları ve oğulları Cogs buluştuğu yer, iyonları ve oğulları uzunluğu ortaya çıkarmak için kas ve doku kesip. </li><li> Benekli hipodermis yama dönün ve kesti. Bu STG çevreleyen arter bir açıklık bırakmanız gerekir. , Iki kaslar bu kanadını STG ayrı delikten kesin. Bu kasların uçları yukarı kaldırın ve sağ kesim, onları arter ayırmak için alns ve agns maruz altında . Stn üzerinde uzanan kıkırdak olarak kadarıyla yan kasları ayırın. </li><li> Mvns, dgn STG aşağı çevreleyen arter doku kesilip, dvn karşılamak. Lvns dvn Çalışma ve lvns kaplayan beyaz, narin doku kesti. </li><li> Psn hazırlanması alt yakın kemik parçası üzerinde bulun. Psn LVN ve dlvn kavşak bulmak için kullanılabilir. </li><li> Sonraki pyn ve PDN bulmak için dlvn izleyin. Pyn sık sık açılması pilorik ampulla üzerine sarar. PDN dlvn çatal kapalı ve pilorik ampulla ve kardiyo pilorik valf kasları arasındadır. Hazırlık net Sylgard çanak transfer edildiğinde kolayca karışabilir, çünkü bu sinirlerin bağlı bazı kas bırakın. </li><li> Bir kez tüm sinirlerin STNS ve mide, doku ve istenmeyen sinirler arasında kalan herhangi bir bağlantıları, sever ortaya edilmiştir. Dikkatli bir şekilde, herhangi bir cevapsız bağlantıları kesme, mide geri kalanı uzakta STNS hareket. </li><li> Sylgard artık çok yapışkan ya da kuru hissedene kadar Sylgard yüzey üzerinde sürtünme mide dokusu kalan kitle ile açık Sylgard çanak durumu. Sylgard hidrofobik ve Sylgard kaplı çanak bu şekilde şartlandırılmış değilse STNS kuvvetle uyacaktır. </li><li> Çanak bazı soğuk tuzlu su ekleyin. Forseps ile commissural sinirlerin üst ucunu alın ve STNS açık Sylgard çanak getirmek. </li><li> STNS Sylgard üzerine sabitleyin. Commissural sinirler beyin Sever ve Minuten pimleri Cogs dört ucu aşağı ilk sabitlemek için kullanabilirsiniz. Hazırlık IVN Sylgard uzak işaret olup olmadığını kontrol sağ tarafında olduğunu emin olun. STG sağ taraftaki dgn STG altında biraz çıkmalısınız. Sinirin geri kalanı Pin aşağı ince tel iğneler ile sona erer. </li><li> STNS kalan kas veya doku temizleyin. </li><li> Desheath bir iğne tutucu ve biraz bağladım, ince, tungsten iğne kullanarak STG. STG kılıf hücre gövdeleri uzak bir köşesinde küçük bir delik açın ve bu delik kullanmak kılıf katmanları arasındaki almak için. STG neuropil ve hücre organları açığa çıkarmak için ayrılmış bir kılıf kapağı dikkatlice kesin. </li><li> Hücre gövdeleri erişilebilirlik artırmak ve hücre içi kayıtları için STG stabilize etmek için aşağı kılıfı herhangi bir flep Pin. Her sinir gergin ve ekstraselüler bir kayıt (bkz. Şekil 3) aralıklı sağlanması STNS geri kalanı Re-pin. </li></ol> 3. Sonuçlar <ol><li> İdeal olan, tüm sinirlerin özellikle çizik ve hasar, kaydedilmiş olacak olanlar ücretsiz olmalıdır. Sinirlerin Yok karışık veya bükülmüş olmalıdır. STG neuropil etrafında bir sakal oluşumu düzenlenen tüm hücrelerin sağlam olmalıdır. Sağlam STNS bilateral simetrik ve bacaklar, kollar ve kafa olarak ön uç olarak mvns lvns bir minyatür gibi görünüyor. </li></ol> Kısaltmalar: <table border="1"><tbody><tr><td> STNS </td><td> Stomatogastric sinir sistemi </td></tr><tr><td> STG </td><td> Stomatogastric ganglion </td></tr><tr><td> COG </td><td> Commissural ganglion </td></tr><tr><td> RG </td><td> Özofagus ganglion </td></tr><tr><td> mvn </td><td> Ortalama ventriküler sinir </td></tr><tr><td> iyon </td><td> İnferior özofagus sinir </td></tr><tr><td> oğlum </td><td> Üstün özofagus sinir </td></tr><tr><td> AGN </td><td> Ön mide sinir </td></tr><tr><td> Aln </td><td> Anterior lateral sinir </td></tr><tr><td> dgn </td><td> Dorsal mide sinir </td></tr><tr><td> dvn </td><td> Dorsal ventriküler sinir </td></tr><tr><td> LVN </td><td> Lateral ventrikül sinir </td></tr><tr><td> psn </td><td> Arka mide sinir </td></tr><tr><td> pyn </td><td> Pilorik sinir </td></tr><tr><td> PDN </td><td> Pilorik dilatör sinir </td></tr><tr><td> dlvn </td><td> Lateral ventrikül sinirin dorsal dalı </td></tr><tr><td> IVN </td><td> İnferior ventriküler sinir </td></tr></tbody></table><img src="/files/ftp_upload/1207/figure_1_90ppi.jpg" alt= &quot;Şekil 1&quot; /&gt; Şekil 1: diseksiyon önce mide STNS yaklaşık yerleştirme gösteren diyagram. <img src="/files/ftp_upload/1207/figure_2_90ppi.jpg" alt="Şekil 2" /> Şekil 2:, açık yeşil, mide ve STNS bir yerleşim alt kısmı çeşitli kasların Diyagramı . STG her hücre tipi kasları innerve bilinen listelenir. Bu harita, planlama, belirli bir hücre (nezaket Nadim laboratuar) bir sinyal taşıyan sinir incelemek yararlı olacaktır. <img src="/files/ftp_upload/old/1207_2009_1_12_5/figure_3.jpg" alt="Şekil 3" /> Şekil 3: STNS, hücre içi elektrotlar ve hücre dışı kuyu (Marder ve Bucher, 2007) ile şematik .

<ol>
	<li>Harris-Warrick, R. M., Marder, E., Selverston, A. I., Moulins, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Dynamic Biological Networks: The stomatogastric nervous system. , (1992).</li><li>Marder, E., Bucher, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+circuit+dynamics+using+the+stomatogastric+nervous+system+of+lobsters+and+crabs.">Understanding circuit dynamics using the stomatogastric nervous system of lobsters and crabs.</a> Annu. Rev. Physiol. 69, 291-316 (2007).</li><li>Maynard, D. M., Dando, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+stomatogastric+neuromuscular+system+in+Callinectes+sapidus,+Homarus+Americanus+and+Panulirus+argus+(Decapoda+crustacean).">The structure of the stomatogastric neuromuscular system in Callinectes sapidus, Homarus Americanus and Panulirus argus (Decapoda crustacean).</a> Philos. Trans. R. Soc. Lond. B Biol. Sci. 268 (892), 161-220 (1974).</li><li>Selverston, A. I., Russell, D. F., Miller, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Stomatogastric+Nervous+System:+Structure+and+function+of+a+small+neural+network.">The Stomatogastric Nervous System: Structure and function of a small neural network.</a> Prog. Neurobiology. 7, 215-290 (1976).</li></ol>

STNS diseksiyonu bir elektrofizyoloji veya immünohistokimya deney, ya da hücre hücre kültürü elde etmek için yerine ilk adımdır. Amaçlanan deney ne olursa olsun, brüt diseksiyon değişmeden kalır. Ancak, ince diseksiyon değişebilir. Öncesinde kayıt hücreleri ve sinirler zaman planı için önemlidir. STG nöronların çoğu mide en az bir kas innerve (bkz. Şekil 2) ve innerve hedef göre sınıflandırılır süreçleri var. Bu nedenle, ekstraselüler kayıtları tam intraselüler kaydedilmiş olan bi...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Black Sylgard 170</td>
<td>Other</td>
<td>Dow Corning</td>
<td>170</td>
<td>Gross Dissection</td>
</tr>
<tr>
<td>Fine Scissors</td>
<td>Surgery</td>
<td>FST</td>
<td>14090-09</td>
<td>&nbsp;</td>
<tr>
<td>Insect Pins size 6</td>
<td>Surgery</td>
<td>FST</td>
<td>26000-65</td>
<td>&nbsp;</td>
<tr>
<td>Mayo Scissors</td>
<td>Surgery</td>
<td>FST</td>
<td>14110-17</td>
<td>&nbsp;</td>
<tr>
<td>Micro-Spatula</td>
<td>Surgery</td>
<td>Ward Sci.</td>
<td>15 V 4313</td>
<td>&nbsp;</td>
<tr>
<td>Rongeurs</td>
<td>Surgery</td>
<td>FST</td>
<td>16000-14</td>
<td>&nbsp;</td>
<tr>
<td>Tissue Forceps</td>
<td>Surgery</td>
<td>FST</td>
<td>11021-12</td>
<td>&nbsp;</td>
<tr>
<td>Fine Tungsten Wire (0.001 in. diameter)</td>
<td>Surgery</td>
<td>California Fine Wire</td>
<td>&nbsp;</td>
<td>Fine Dissection</td>
</tr>
<tr>
<td>Forceps #5</td>
<td>Surgery</td>
<td>FST</td>
<td>91150-20</td>
<td>&nbsp;</td>
<tr>
<td>Minuten Pins</td>
<td>Surgery</td>
<td>FST</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Moria Spring Scissors (7mm blades)</td>
<td>Surgery</td>
<td>FST</td>
<td>15370-52</td>
<td>&nbsp;</td>
<tr>
<td>Petri Dish (100x15mm)</td>
<td>Other</td>
<td>Fischer	</td>
<td>08-757-12</td>
<td>&nbsp;</td>
<tr>
<td>Pin Holder</td>
<td>Surgery</td>
<td>FST</td>
<td>26018-17</td>
<td>&nbsp;</td>
<tr>
<td>Super fine forceps #5SF</td>
<td>Surgery</td>
<td>FST</td>
<td>11252-00</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 182</td>
<td>Other</td>
<td>Dow Corning</td>
<td>182</td>
<td>&nbsp;</td>
<tr>
<td>Tungsten Needles</td>
<td>Surgery</td>
<td>FST</td>
<td>10130-05</td>
<td>&nbsp;</td>
<tr>
<td>Dissecting Microscope</td>
<td>Microscope</td>
<td>Wild</td>
<td>MC3</td>
<td>Misc.</td>
</tr>
<tr>
<td>Jonah Crab (C. borealis)</td>
<td>Animal</td>
<td>Commercial Lobster</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>
			<div>
						
		</div>

cancer borealis stomatogastric nervous system dissection

Iyi karakterize bir hücre nispeten az sayıda (yaklaşık 25 C borealis) içerdiğinden stomatogastric ganglion (STG), hücresel ve ağ etkileşimleri çalışmak için mükemmel bir model . STG hücreler çıkışları geniş bir sergi ve mide motor eylemler için sorumludur. Mide, üç iç dişleri ile gıda ve gıda midgut ulaşmadan önce filtreler pyloru parçalayan gastrik değirmen içerir. STG merkezi desen jeneratörleri (CPG&#39;lerin hepsi aynı) olarak bilinen mide değirmen ve pyloru kontrol etmek için iki ritmik çıkışları üretir. STG Her hücre bu ritimleri birini veya her ikisini katılabilir. Bu CPG&#39;lerin nöromodülasyon çalışmada, homeostazı, hücresel ve ağ değişkenlik, ağ geliştirme ve ağ kurtarma için izin verir. Jonah yengeç (Kanser borealis) stomatogastric sinir sistemi (STNS) diseksiyonu iki parça halinde yapılır; kaba ve ince diseksiyon. Brüt diseksiyon tüm mide yengeç disseke edilir. Ince diseksiyon sırasında STNS mide bir diseksiyon mikroskobu ve mikro diseksiyon araçları (bkz. Şekil 1) kullanılarak elde edilir. STNS STG, özofagus ganglion (RG) ve commissural ganglionlar (cog) yanı sıra mide kasları innerve sinirleri içerir. Burada, ekstrasellüler kayıtları için kullanılan olurdu STG hücrelerin intraselüler ve periferik sinirleri kaydedilen olacak bir elektrofizyoloji deney için hazırlık STNS tam bir diseksiyon nasıl göstermektedir. Somata ve neuropil ortaya çıkarmak için ganglion desheathing bizim teknik olarak istenilen sinirler bulmak için uygun tekniği de gösterilmiştir.

Watch this Scientific Journal Video about Cancer Borealis Stomatogastric Nervous System Dissection at JoVE.com

Cancer Borealis Stomatogastric Nervous System Dissection

Jonah yengeç stomatogastric sinir sistemi (STNS) (<em&gt; C. poyraz</em&gt; Elektrofizyoloji, immünhistokimya ve hücre kültürü çalışmaları için kullanılabilir). STNS çıkarma iki bölümden yapılır: kaba ve ince diseksiyon.

Kanser Borealis Stomatogastric Sinir Sistemi Diseksiyon

The stomatogastric ganglion (STG) is an excellent model for studying cellular and network interactions because it contains a relatively small number of ...

cancer-borealis-stomatogastric-nervous-system-dissection

Research

JoVE Journal

Biology

Cancer Borealis Stomatogastric Nervous System Dissection

12.5K Görüntüleme.  Brandeis. Jonah yengeç stomatogastric sinir sistemi (STNS) ( C. poyraz Elektrofizyoloji, immünhistokimya ve hücre kültürü çalışmaları için kullanılabilir). STNS çıkarma iki bölümden yapılır: kaba ve ince diseksiyon.

Video: Cancer Borealis Stomatogastric Nervous System Dissection

Dissection of Drosophila Ovaries

Drosophila Yumurtalıkların diseksiyonu

Biyoloji

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Drosophila melanogaster Larvaların MSS diseksiyonu

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPC Fare Embriyolar diseksiyonu

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

Isolation of Mononuclear Cells from the Central Nervous System of Rats with EAE

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the immune response to an infection of the CNS, such as poliomyelitis, Lyme neuroborreliosis, or neurosyphilis, it is often necessary to isolate the CNS-infiltrating immune cells.
In this video-protocol we demonstrate how to isolate mononuclear cells (MNCs) from the CNS of a rat with EAE. The first step of this procedure requires a cardiac perfusion of the rodent with a saline solution to ensure that no blood remains in the blood vessels irrigating the CNS. Any blood contamination will artificially increase the number of apparent CNS-infiltrating MNCs and may alter the apparent composition of the immune infiltrate. We then demonstrate how to remove the brain and spinal cord of the rat for subsequent dilaceration to prepare a single-cell suspension. This suspension is separated on a two-layer Percoll gradient to isolate the MNCs. After washing, these cells are then ready to undergo any required procedure. 
Mononuclear cells isolated using this procedure are viable and can be used for electrophysiology, flow cytometry (FACS), or biochemistry. If the technique is performed under sterile conditions (using sterile instruments in a tissue culture hood) the cells can also be grown in tissue culture medium. A given cell population can be further purified using either magnetic separation procedures or a FACS.

In this video we demonstrate how to isolate mononuclear cells from the central nervous system of rats with experimental autoimmune encephalomyelitis.

EAE Ratlarda Merkez Sinir Sistemi Mononükleer Hücreleri İzolasyon

Whether studying an autoimmune disease directed to the central nervous system (CNS), such as experimental autoimmune encephalomyelitis (EAE, 1), or the ...

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila Larvaların NMJ Diseksiyon

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Diseksiyonu Saccharomyces cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Homarus Americanus Stomatogastric Nervous System Dissection

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the activity of interest and are accessible and amenable to experimental methods. The stomatogastric nervous system (STNS) of the American lobster (Homarus americanus; also know was the Atlantic or Maine lobster) has been established as a model system for studying rhythm generating networks and neuromodulation of networks. The STNS consists of 3 anterior ganglia (2 commissural ganglia and an oesophageal ganglion), containing modulatory neurons that project centrally to the stomatogastric ganglion (STG). The STG contains approximately 30 neurons that comprise two central pattern generating networks, the pyloric and gastric networks that underlie feeding behaviors in crustaceans1,2. While it is possible to study this system in vivo3, the STNS continues to produce its rhythmic activity when isolated in vitro. Physical isolation of the STNS in a dish allows for easy access to the somata in the ganglia for intracellular electrophysiological recordings and to the nerves of the STNS for extracellular recordings. Isolating the STNS is a two-part process. The first part, dissecting the stomach from the animal, is described in an accompanying video article4. In this video article, fine dissection techniques are used to isolate the STNS from the stomach. This procedure results in a nervous system preparation that is available for electrophysiological recordings.

Homarus Americanus Stomatogastric Nervous System Dissection

We describe the fine dissection of the stomatogastric nervous system from the stomach of the American lobster (Homarus americanus).

Homarus americanus Stomatogastric Sinir Sistemi Diseksiyon

With the goal of understanding how nervous systems produce activity and respond to the environment, neuroscientists turn to model systems that exhibit the ...

Deficient Pms2, ERCC1, Ku86, CcOI in Field Defects During Progression to Colon Cancer

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other malignancies of the same tissue type, often in a nearby location. Such field defects have been indicated in colon cancer. The molecular abnormalities that are responsible for a field defect in the colon should be detectable at high frequency in the histologically normal tissue surrounding a colonic adenocarcinoma or surrounding an adenoma with advanced neoplasia (well on the way to a colon cancer), but at low frequency in the colonic mucosa from patients without colonic neoplasia.
Using immunohistochemistry, entire crypts within 10 cm on each side of colonic adenocarcinomas or advanced colonic neoplasias were found to be frequently reduced or absent in expression for two DNA repair proteins, Pms2 and/or ERCC1. Pms2 is a dual role protein, active in DNA mismatch repair as well as needed in apoptosis of cells with excess DNA damage. ERCC1 is active in DNA nucleotide excision repair. The reduced or absent expression of both ERCC1 and Pms2 would create cells with both increased ability to survive (apoptosis resistance) and increased level of mutability. The reduced or absent expression of both ERCC1 and Pms2 is likely an early step in progression to colon cancer.
DNA repair gene Ku86 (active in DNA non-homologous end joining) and Cytochrome c Oxidase Subunit I (involved in apoptosis) had each been reported to be decreased in expression in mucosal areas close to colon cancers. However, immunohistochemical evaluation of their levels of expression showed only low to modest frequencies of crypts to be deficient in their expression in a field defect surrounding colon cancer or surrounding advanced colonic neoplasia.
We show, here, our method of evaluation of crypts for expression of ERCC1, Pms2, Ku86 and CcOI. We show that frequency of entire crypts deficient for Pms2 and ERCC1 is often as great as 70% to 95% in 20 cm long areas surrounding a colonic neoplasia, while frequency of crypts deficient in Ku86 has a median value of 2% and frequency of crypts deficient in CcOI has a median value of 16% in these areas. The entire colon is 150 cm long (about 5 feet) and has about 10 million crypts in its mucosal layer. The defect in Pms2 and ERCC1 surrounding a colon cancer thus may include 1 million crypts. It is from a defective crypt that colon cancer arises.

Reduced/absent expression of Pms2 and/or ERCC1 in entire crypts is a frequent event within 10 cm on each side of colonic adenocarcinomas, likely the basis of a field defect with high mutability and progression to cancer. Deficiency in Ku86 or CcOI is much less frequent in these field defects.

Eksik Pms2 ERCC1, Ku86 Kolon Kanseri ilerleme sırasında Alan Kusur CcOI

In carcinogenesis, the "field defect" is recognized clinically because of the high propensity of survivors of certain cancers to develop other ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Yetişkin gelen Oenocytes diseksiyonu Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Yetişkin Zebra balığı Böbrek diseksiyonu

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...