Bu çalışma, Tarım ve Tarım-Gıda Kanada A-Baz projesi &quot;işareti destekli seçiminde çok değişkenli kalite özellikleri için Phenomic boşluğu azaltmak için et örnekler için yüksek verimli tekniklerinin geliştirilmesi&quot; bir parçasıydı. Sığır Birimi ve Lacombe Araştırma Merkezi&#39;nde Et İşleme personel yetenekli yardım içtenlikle takdir edilmektedir. Yazarlar ayrıca Christine Burbidge-Boyd, Fran Costello, Glynnis Croken ve Rhona Thacker en özel teknik yardım teşekkür etmek istiyorum.

1. Steak Koleksiyon <ol><li> 24 saatte aşağıdaki sınıflandırma - 48 saat sonrası kesim, longissimus kas (ya da göğüs ve / veya lumborum sonu) sığır karkas kaldırılır ve kalite analizi için toplanır. </li><li> Kas deri altı yağ ve kare kapalı kesilmiş. Kas sonra güvenliği ve gıda işleme protokolleri takip 2.5 cm (1) biftek halinde kesilir. Bireysel biftek kesme sonrasında, uygun bir etiket (hayvan numarası, yan, kas konum veya süre) ilgili biftek yerleştirilir. </li><li> Biftek sonra depolama için hazırlanır. Taze kalması, bunlar bir tepsi yerleştirilir ve ızgara alınır. Taze biftek hemen pişmiş olamaz, bunlar yemek su kaybını önlemek için, başlayabilirsiniz kadar plastik örtülü olması gereken. Biftek bir soğutucu yaş, ya da donmuş edilecekse, ağırlık bir oksijen geçirimsiz vakum paket ba konulmadan önce iki ondalık basamağa kaydedilirg ve mühürlü bir vakum paketleme makinesi kullanarak. Biftek daha sonra kendi uzun süreli depolama konumlarına yerleştirilir. </li></ol> 2. Yemek Prosedürü <ol><li> Örnek dondurulmuş ise, açmak için bir gecede 4 ° C soğutucu dondurucu ve yerden çıkarın. </li><li> Pişirme gününde, 210 ön ısıtma için bir açık ocak ızgarası ° C örnek pişirme ve tarama cihazının çıkış içine ısıl sokarak sıcaklık kayıt cihazı sistem ve bilgisayar hazırlar. Hafifçe yağlayın yemek başlamadan önce sebze yaklaşık 10 dakika kısaltarak ile ızgara. </li><li> Pişirmek için başlamadan önce, soğutucudan biftek çıkarın. Ambalajından örnek çıkarın ve kağıt havlu ile biftek aşırı nem kurulayın. Iki ondalık basamağa biftek ham ağırlık kaydedin. </li><li> Uzunlamasına ekseni boyunca orta biftek içine bir termokupl yerleştirin. Pişirme verilerini kaydetmek için termokupl tarama başlatın. </li><li> Yerleştirinızgara biftek, biftek ve ızgara düz pişirme yüzeyi için termokupl paralel tutarak. Prob paralel tutulması değilse, bu yanlış ısı ölçümü neden olabilir. </li><li> 35.5 arasında bir iç sıcaklığa biftek yemek ° C, daha sonra numunenin üzerine çevirmek ve 71 son bir iç sıcaklığa pişirme devam ° C Iç sıcaklığı 71 ° C olduğunda, ızgara örnek çıkarın ve biftek termokupl çekin. </li><li> Bir yemek kilo isteniyorsa, biraz soğumasını biftek (1-2 dk), daha sonra aşırı nemi ve iki ondalık basamağa örnek tartmak için bir kağıt havlu ile örnek leke sağlar. </li><li> Pişirme veri gerekiyorsa, termokupl tarama verileri kaydetmek. </li></ol> 3. Açı Ayarlama ve Örnek Hazırlama <ol><li> Bir kare sonu oluşturmak için biftek yan ucundan 2 cm - örnek hala ızgara sıcakken, bir kesim 1 yapmak. </li><li> Wid genelinde bir 5 cm bölümübiftek paralelin orijinal kesim için, 5 cm bir bıçak çentik ile bir &#39;örnek boyutlandırma kutu&#39; kullanılarak hazırlanmıştır. </li><li> Kas lifi açı yönü biftek bıçak kesme yüzeyi üzerinde bir iletki yerleştirerek ölçülebilir. </li><li> Değişken açılı kesme kutusu çift kanatlı bıçak yol için alt delikli özel alt metal plaka kaydırarak numunenin ilgili açısına ayarlanır. Ilgili açıyla alt metal plaka sabitlemek için kolları sıkın. </li><li> Kas lifi yönünde çift kanatlı bıçak açısını paralel olacak şekilde değişken açılı kesme kutusunun kesim alanına biftek 5 cm bölümüne yerleştirin. </li><li> Çift kanatlı bıçak bıçak ilgili açıyla tüm örnek geçecek sağlamak böylece, daha önce kesilmiş 5 cm parça genelinde bir 1 cm dilim kesmek için kullanılır. Biftek yerleştirirken, bu en yakın kesim alanının kenarına dokunmadan emin olunKullanıcı, kesme hareketi olarak kullanıcıya karşı biftek çeker. </li><li> , Kesim kesim kutusuna çift kanatlı bıçak yerleştirin ve 1 cm şerit halinde et kesmek için bıçak çekin. Kullanıcıya karşı bıçak çekerken et yırtılmasını önlemek için, hafif bir kesme hareketi kullanın. Kılavuz plakası biri kenarına bıçak bıçak eşit kesme oranlarını korumak için tutun. </li><li> Sonuç olarak, liflerin sürekli dilim paralel olarak çalışan, 5 cm uzunluğunda bir sıcak, 1 cm kalınlığında bir dilim olmalıdır. Bu dilim sonra et doku dilim kesme kuvvet analizi için kullanılır. </li></ol> 4. Kesme Kuvvet Analizi <ol><li> Kesme kuvveti analizi belirlemek için, dilim kesme kuvveti protokolü (SSF) değişken açılı kesme kutusu ile elde dilim 5 cm 1 cm kullanır. Düz kesme dilim bıçak ile bir doku analizörü makine kullanarak, analiz sıcak dilim yapılır. </li><li> 50 kg yük hücresi ve set ile Doku Analyzer hazırlayın500 mm / dak &#39;lık bir çapraz kafa hızı ile 48 mm arasında, ve kesme mesafesi. </li><li> Kesme kesme dilim lifler için gerekli olan maksimum gücü doğru bir analiz sağlamaktır kas lifleri dik olarak, bir kez gerçekleştirilir. </li><li> Dilim merkezi aracılığıyla tamamen makaslanmış sonra, kaydetmek ve veri kaydı. </li></ol>

<ol>
	<li>Jayasooriya, S. D., Torley, P. J., D'Arcy, B. R., Bhandari, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+high+power+ultrasound+and+ageing+on+the+physical+properties+of+bovine+Semitendinosus+and+Longissimus+muscles.">Effect of high power ultrasound and ageing on the physical properties of bovine Semitendinosus and Longissimus muscles.</a> Meat Science. 75, 628-639 (2007).</li><li>Koohmaraie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+proteinases+and+meat+aging.">Muscle proteinases and meat aging.</a> Meat Science. 36, 93-104 (1994).</li><li>Lepetit, J., Culioli, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+of+meat.">Mechanical properties of meat.</a> Meat Science. 36, 203-237 (1994).</li><li>Honikel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reference+methods+supported+by+OECD+and+their+use+in+Mediterranean+meat+products.">Reference methods supported by OECD and their use in Mediterranean meat products.</a> Food Chemistry. 59, 573-582 (1997).</li><li>Monin, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+methods+for+predicting+quality+of+whole+meat.">Recent methods for predicting quality of whole meat.</a> Meat Science. 49, S231-S243 (1998).</li><li>Shackelford, S., Wheeler, T., Koohmaraie, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+slice+shear+force+as+an+objective+method+of+assessing+beef+longissimus+tenderness.">Evaluation of slice shear force as an objective method of assessing beef longissimus tenderness.</a> Journal of Animal Science. 77, 2693-2699 (1999).</li><li>Ju&#225;rez, M., Hui, Y. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beef+Texture+and+Juiciness.">Beef Texture and Juiciness.</a> Handbook of meat and meat processing. , 177-206 (2012).</li><li>Kerth, C. R., Montgomery, J. L., Lansdell, J. L., Ramsey, C. B., Miller, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shear+gradient+in+longissimus+steaks.">Shear gradient in longissimus steaks.</a> Journal of Animal Science. 80, 2390-2395 (2002).</li><li>Janz, J. A. M., Aalhus, J. L., Dugan, M. E. R., Price, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mapping+method+for+the+description+of+Warner-Bratzler+shear+force+gradients+in+beef+Longissimus+thoracis+et+lumborum+and+Semitendinosus.">A mapping method for the description of Warner-Bratzler shear force gradients in beef Longissimus thoracis et lumborum and Semitendinosus.</a> Meat Science. 72, 79-90 (2006).</li><li>Derington, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relationships+of+slice+shear+force+and+Warner-Bratzler+shear+force+of+beef+strip+loin+steaks+as+related+to+the+tenderness+gradient+of+the+strip+loin.">Relationships of slice shear force and Warner-Bratzler shear force of beef strip loin steaks as related to the tenderness gradient of the strip loin.</a> Meat Science. 88, 203-208 (2011).</li><li>Shackelford, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slice+Shear+Force.">Slice Shear Force.</a> Beef Facts. , (2009).</li><li>Henrickson, R. L., Mjoseth, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tenderness+variation+in+two+bovine+muscles.">Tenderness variation in two bovine muscles.</a> Journal of Animal Science. 23, 325-331 (1964).</li><li>Ju&#225;rez, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+relative+contribution+of+ante-+and+post-mortem+factors+to+the+variability+in+beef+texture.">Quantifying the relative contribution of ante- and post-mortem factors to the variability in beef texture.</a> Animal. FirstView, 1-10 (2012).</li><li>Shanks, B. C., Wulf, D. M., Maddock, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+note:+The+effect+of+freezing+on+Warner-Bratzler+shear+force+values+of+beef+longissimus+steaks+across+several+postmortem+aging+periods.">Technical note: The effect of freezing on Warner-Bratzler shear force values of beef longissimus steaks across several postmortem aging periods.</a> Journal of Animal Science. 80, 2122-2125 (2002).</li><li>Lawrence, T. E., King, D. A., Obuz, E., Yancey, E. J., Dikeman, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+electric+belt+grill,+forced-air+convection+oven,+and+electric+broiler+cookery+methods+for+beef+tenderness+research.">Evaluation of electric belt grill, forced-air convection oven, and electric broiler cookery methods for beef tenderness research.</a> Meat Science. 58, 239-246 (2001).</li><li>Aalhus, J., Ju&#225;rez, M., Aldai, N., Uttaro, B., Dugan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meat+preparation+and+eating+quality.">Meat preparation and eating quality.</a> , 1058-1063 (2009).</li><li>Sa&#241;udo, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+slaughter+weight,+breed+type+and+ageing+time+on+beef+meat+quality+using+two+different+texture+devices.">The effects of slaughter weight, breed type and ageing time on beef meat quality using two different texture devices.</a> Meat Science. 66, 925-932 (2004).</li></ol>

Biz ifşa hiçbir şey yok.

Açısı ayarlanabilir kutusu SSF bıçak her zaman yerine sabit 45 ° açılı kesim 8 ile olabilirdi yaklaşık olarak dik daha, gerçek bir dik açı ile kas lifleri makası sağlar. Değişken açılı kesme kutusunun Uygulama numunedeki maksimum kesme kuvveti daha doğru tasvirleri için izin verir, ve değişken açılı kesme kutusu geliştirilmiştir dair objektif bir kalite analizi teknikleri iyileştirilmesi bu temel üzerinde. Ayrıca, ayarlanabilir açı kutusu (30 ° ila 60 °) tarafından sunula...

Hassasiyet et 1 en önemli kalite özelliklerinden biridir. Sığır hassasiyet tutarsızlık sığır eti endüstrisinin 2 karşılaştığı önemli sorunlardan biri olarak tespit edilmiştir. Bu tartışmasız en iyi göstermiştir etkili yöntem olarak bir tam işlenmiş kesme levha bir üçgen delik ile karakterize Warner-Bratzler kesme kuvveti (WBSF) testi,,, et duyusal hassasiyet 3,4 belirtmek için kullanılan en yaygın yöntemdir et tokluk 5 için duyusal paneli puanları ile korelasyon. Bununla birlikte, dilim kesme kuvveti protokolü (SSF) standart WBSF protokol 7 için bir alternatif olarak, kas dokusu ve hassasiyet 6 analiz etmek için önemli bir teknik olmuştur. Bu hızlı analizi veya numune, yüksek bir sayı işlenmesi gereken durumlarda faydalıdır. Hala sıcakken SSF için, sadece bir çekirdek biftek alınır, karşı çoklu çekirdek (3-6), genellikle kıç biftek alınanWBSF 6 soğutma er 24 saat. Bu hassasiyet ortalaması WBSF için en iyi temsil sahip biftek merkezi ve orta kısmı ile, biftek 9 için Medial taraf arasında değiştiği tespit edilmiştir, bu bir dilim kaynaktan, SSF, bütün biftek 8 arasında ortalama doku analiz . SSF için düşüşünü bir biftek içinde, lateralden medial kenarlarına, kesme kuvveti değerleri 9 değişik olduğu, bu, ancak, sürekli 5 cm bölümleri yapmak için boyutlandırma kutusunu kullanarak, bu gelebilir değişkenliği azaltabilir biftek farklı kesimleri kullanarak. Bazı biftek farklı boyutlarda olduğundan Ancak, 5 cm dilim sonra kesme kuvveti değerleri 10 etkileyebilecek biftek farklı yerlerde, oluşabilir. Öte yandan, SSF protokolde analiz edilecek bölüm elde etmek için tasarlanan orijinal kesim kutusu sadece 2 sabit açılı, 45 ° ve 90 ° sağlar, ancak kasbiftek içinde ve kas 11 içinde elyaf oryantasyonu değişir. Shackelford ve Wheeler 12 ortalama longissimus açısı 43.8 yakın 45 oldu °, ° ve örnekleri her zaman aynı yerde toplanmıştır zaman bu nedenle uygun görülen olduğunu belirtti. Bununla birlikte, Derington ve ark. 33.1 ° 11 ve 53.9 ° arasında longissimus kas boyunca liflerin açısındaki bir dizi Min. Böylece, zaman, araştırma amacıyla, aynı hayvan birkaç biftek 45 ° veya 90 ° potansiyel doğruluğu azaltır ya tüm fiber açıları atama, analiz edilmesi gerekir. Kesme açısı ölçmek ve ayarlamak için kapasitesine sahip dilim için sürekli kas lifleri paralel çalışmasını sağlar gibi bir değişken açılı kesme kutusunun kullanımı, maksimum kesme kuvveti daha doğru bir tasviri sağlayabilir.

<table border="1">
	<tbody>
		<tr>
			<td>Name of equipment</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Vacuum Packager</td>
			<td>Koch Equipment, Kansas City, MO, USA</td>
			<td>Model UV2100</td>
		</tr>
		<tr>
			<td>Vacuum bags</td>
			<td>Winpak Ltd., Winnipeg, MB, Canada</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Thermocouples and Scanning Device</td>
			<td>Agilent / Hewlett Packard, Santa Clara, CA, USA</td>
			<td>34970A Data Acquisition Switch Unit</td>
		</tr>
		<tr>
			<td>Grill</td>
			<td>Garland Commercial Ranges Ltd., Mississauga, ON, Canada</td>
			<td>Model ED-30-B</td>
		</tr>
		<tr>
			<td>Crisco Vegetable Shortening</td>
			<td>The J.M. Smucker Company, Orrville, OH, USA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Sample sizing box</td>
			<td>G-R Manufacturing Co., Manhattan, KS, USA</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Angle adjustable box</td>
			<td>Innovation Centre, Red Deer College, Red Deer, AB, Canada</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Texture Analyzer Machine</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>Model TA-XT Plus</td>
		</tr>
		<tr>
			<td>Load Cell, 50 kg</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-XT Plus</td>
		</tr>
		<tr>
			<td>USDA Warner-Bratzler knife w/guillotine block</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-7</td>
		</tr>
		<tr>
			<td>Flat rectangle Blade for Slice Shear Force</td>
			<td>Texture Technologies, Hamilton, MA, USA</td>
			<td>TA-7C</td>
		</tr>
	</tbody>
</table>

introducing an angle adjustable cutting box for analyzing slice shear force in meat

Araştırma longissimus kas lifi açısı biftek içinde ve kas boyunca yere bağlı olarak, farklı olabileceği anlamına gelir. Bunun yerine orijinal sabit 45 ° veya 90 ° &#39;lik kesme kuvveti test etmek için kesme açısı kullanmanın bıçak açılarının her numunenin lif açısına karşılık gelecek şekilde, değişken açılı kesim kutusu ayarlanabilir. 2 dakika içinde 71 bir iç sıcaklığı yemek sonra ° 210 olarak belirlendi açık ocak ızgara C ° C, 5 cm çekirdek bir 1 cm 2 bıçak bıçak kullanarak, kas lifi yönüne paralel, biftek kesilir 1 set ayrı cm. Bu sıcak çekirdek daha sonra et doku değerlendirmek için Dilim Kesme Kuvveti protokolü (SSF) tabi tutulur. Değişken açılı kesme kutusu ve SSF protokolünün kullanımı dilim ve kas lifleri sürekli paralel olarak, maksimum kesme kuvveti doğru bir temsilini sağlar. Bu nedenle, değişken açılı kesim kutusuna SSF protokolü ile bağlantılı olarak, yüksek inci olarak kullanılabilirroughput tekniği doğru diğer kaslarda, potansiyel, longissimus kas farklı yerlerde et hassasiyet değerlendirmek ve.

Otuz bir bitmiş ticari sığır steers katledildi ve gövde (528-601 kg) ayrıldı. Sol tarafta 2 ° C&#39;de 3 saat karıştırıldı ve daha sonra 10 ° C &#39;de tutulurken hassasiyet değişkenlik oluşturmak için, sağ tarafı, hemen 2 ° C&#39; de muhafaza edilmiştir Sağ ve sol taraftan longissimus kasları kesim sonrası 24 saat çıkarıldı. Dakikada sol kanattan gelen örnekleri (&quot;İHALE&quot; tedavi) analiz edilmeden önce 6 d yaş iken sağ taraf (&quot;SERT&quot; tedavi) alınan numuneler,...

Watch this Scientific Journal Video about Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat at JoVE.com

Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Dilim kesme kuvveti sığır doku analizi için bir referans yöntemdir. Bir açı ayarlanabilir kesme kutusunu kullanarak araştırma amaçlı doğruluğu artırabilir. Içinde farklı yerlerden sonuçları<em&gt; Longissimus</em&gt; Kas Warner-Bratzler kesme kuvveti metodoloji ve farklı kasları için potansiyeli yüksek uyum ile yüksek korelasyon göstermektedir.

Et Dilim Kesme Kuvveti analiz için Açısı Ayarlanabilir Kesme Kutusu Tanıtımı

Research indicates the fibre angle of the longissimus muscle can vary, depending upon location within a steak and throughout the muscle. Instead of using ...

introducing-an-angle-adjustable-cutting-box-for-analyzing-slice-shear

Research

JoVE Journal

Biology

12.8K Görüntüleme.  Agriculture and Agri-Food Canada. Dilim kesme kuvveti sığır doku analizi için bir referans yöntemdir. Bir açı ayarlanabilir kesme kutusunu kullanarak araştırma amaçlı doğruluğu artırabilir. Içinde farklı yerlerden sonuçları Longissimus Kas Warner-Bratzler kesme kuvveti metodoloji ve farklı kasları için potansiyeli yüksek uyum ile yüksek korelasyon göstermektedir.

Video: Introducing an Angle Adjustable Cutting Box for Analyzing Slice Shear Force in Meat

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

E18 Rat Brain Organotipik Dilim Kültür

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Biyoloji

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

Işık tam hücreli Kayıtlar Retina Dilim Uyarıcı Synaptic Akımlar Uyarılmış

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3&#039;UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

3&#39;UTR muhabiri oluşturur ve bir microRNA kullanarak LightSwitch Lusiferaz Test Sistemi ile İnsan microRNA belirlenmesi Hedefler yapışık hücrelere Mimik

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

A Method for Selecting Structure-switching Aptamers Applied to a Colorimetric Gold Nanoparticle Assay

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health and performance. Nucleic acid aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward small molecule targets requires special design considerations. This work describes modification and critical steps of a method designed to select structure-switching aptamers to small molecule targets. Binding sequences from a DNA library hybridized to complementary DNA capture probes on magnetic beads are separated from nonbinders via a target-induced change in conformation. This method is advantageous because sequences binding the support matrix (beads) will not be further amplified, and it does not require immobilization of the target molecule. However, the melting temperature of the capture probe and library is kept at or slightly above RT, such that sequences that dehybridize based on thermodynamics will also be present in the supernatant solution. This effectively limits the partitioning efficiency (ability to separate target binding sequences from nonbinders), and therefore many selection rounds will be required to remove background sequences. The reported method differs from previous structure-switching aptamer selections due to implementation of negative selection steps, simplified enrichment monitoring, and extension of the length of the capture probe following selection enrichment to provide enhanced stringency. The selected structure-switching aptamers are advantageous in a gold nanoparticle assay platform that reports the presence of a target molecule by the conformational change of the aptamer. The gold nanoparticle assay was applied because it provides a simple, rapid colorimetric readout that is beneficial in a clinical or deployed environment. Design and optimization considerations are presented for the assay as proof-of-principle work in buffer to provide a foundation for further extension of the work toward small molecule biosensing in physiological fluids.

A protocol is provided to select structure-switching aptamers for small molecule targets based on a tunable stringency magnetic bead selection method. Aptamers selected with structure-switching properties are beneficial for assays that require a conformational change to signal the presence of a target, such as the described gold nanoparticle assay.

Bir Kolorimetrik Altın Nanopartikül Testi Uygulanan Yapı-anahtarlama aptamers seçilmesi için bir yöntem

Small molecules provide rich targets for biosensing applications due to their physiological implications as biomarkers of various aspects of human health ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag Analizi - A Yüksek Verimli, Sc2.0 Genotiplendirme için Real Time PCR Testi

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

Design and Development of Aptamer&#8211;Gold Nanoparticle Based Colorimetric Assays for In-the-field Applications

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field applications was examined. Target selective AuNP based color assays have been developed in controlled proof-of-concept laboratory settings. However, these schemes have not been exerted to a point of failure to determine their practical use beyond laboratory settings. This work describes a generic approach to design, develop, and troubleshoot an aptamer-AuNP colorimetric assay for small molecule analytes and using the assay for in-the-field settings. The assay is advantageous because adsorbed aptamers passivate the nanoparticle surfaces and provide a means to reduce and eliminate false positive responses to non-target analytes. Transitioning this system to practical uses required defining not only the shelf-life of the aptamer-AuNP assay, but establishing methods and procedures for extending the long-term storage capabilities. Also, one of the recognized concerns with colorimetric readout is the burden placed on analysts to accurately identify often subtle changes in color. To lessen the responsibility on analysts in the field, a color analysis protocol was designed to perform the color identification duties without the need for performing this task on laboratory grade equipment. The method for creating and testing the data analysis protocol is described. However to understand and influence the design of adsorbed aptamer assays, the interactions associated with the aptamer, target, and AuNPs require further study. The knowledge gained could lead to tailoring aptamers for improved functionality.

The design and development of an aptamer&#8211;gold nanoparticle colorimetric assay for the detection of small molecules for in-the-field applications was examined. Additionally, a smart-device colorimetric application (app) was validated and long-term storage of the assay was established for use in the field.

In-alan uygulamaları için tasarım ve Aptamer-Altın Nanopartikül Tabanlı Kolorimetrik Tahliller Gelişimi

The design and development of an aptamer&#8211;gold nanoparticle (AuNP) colorimetric assay for the detection of small molecules for in-the-field ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

Montaj ve &#39;Kayma Rings&#39; Uygulama: Orbital tek yönlü ve Periyodik Akışkan Akışının ve Kayma Stres için Yeni Bir Endotel Modeli

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

Algılama ve ölçme protein Self-montaj In vivo tarafından akış sitometri

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from ...

Automated Charting of the Visual Space of Housefly Compound Eyes

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands of visual units called ommatidia. Each ommatidium samples the optical information from a small solid angle, with an approximate Gaussian-distributed sensitivity (half-width on the order of 1&#730;) centered around a visual axis. Together, the ommatidia gather the visual information from a nearly panoramic field of view. The spatial distribution of the visual axes thus determines the eye's spatial resolution. Knowledge of the optical organization of a compound eye and its visual acuity is crucial for quantitative studies of neural processing of the visual information. Here we present an automated procedure for mapping a compound eye's visual axes, using an intrinsic, in vivo optical phenomenon, the pseudopupil, and the pupil mechanism of the photoreceptor cells. We outline the optomechanical setup for scanning insect eyes and use experimental results obtained from a housefly, Musca domestica, to illustrate the steps in the measurement procedure.

The protocol here describes the measurement of the spatial organization of the visual axes of housefly eyes, mapped by an automatic device, using the pseudopupil phenomenon and the pupil mechanism of the photoreceptor cells.

Housefly Compound Eyes'ın Görsel Alanının Otomatik Grafiği

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands ...

Collection and Long-Term Maintenance of Leaf-Cutting Ants (Atta) in Laboratory Conditions

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled environments enables an enriched comprehension of their biology that can contribute to applied research. This practice is usually employed in population control studies of species that cause economic loss, such as Atta ants. To cultivate their mutualistic fungus, these leaf-cutting ants collect leaves and for this are considered agricultural pests widely distributed throughout the American continent. They are highly socially organized and inhabit elaborated underground nests composed of a variety of chambers. Their maintenance in a controlled environment depends on a daily routine of several procedures and frequent care that are described here. It starts with the collection of queens during the reproductive season (i.e., nuptial flight), which are then individually transferred to plastic containers. Due to the high mortality rate of queens, a second collection can be carried out about 6 months after the nuptial flight, when incipient nests with developed fungus wad are excavated, hand-picked, and placed in plastic containers. In the laboratory, leaves are daily provided to established colonies, and ant-produced waste is weekly removed along with remaining dry plant material. As the fungus garden keeps growing, colonies are transferred to different types of containers according to the experimental purpose. Leaf-cutting ant colonies are placed in interconnected containers, representing the organizational system with functional chambers built by those insects in nature. This setup is ideal to monitor factors such as waste amount, fungus garden health, and the behavior of workers and queen. Facilitated data collection and more detailed observations are considered the greatest advantage of keeping ant colonies in controlled conditions.

Collection and Long-Term Maintenance of Leaf-Cutting Ants Atta in Laboratory Conditions

Here, a protocol is described to successfully collect and maintain healthy Atta (Hymenoptera: Formicidae) ant colonies in laboratory conditions. Additionally, different nest types and configurations are detailed together with possible experimental procedures.

Yaprak Kesici Karıncaların (Atta) Laboratuvar Koşullarında Toplanması ve Uzun Süreli Bakımı

Ants are one of the most biodiverse groups of animals on the planet and inhabit different environments. The maintenance of ant colonies in controlled ...