著者らは、撮影時の彼の助けニコールM.ネメト（ジョージア大学）と鶏の組織を収穫で彼らの助けのためのジャンヌ·M·フェア（LANL）と、スティーブンA.スプリンガーに感謝したいと思います。この研究ではすべての鳥の世話は、ロスアラモス国立セキュリティ、LLCは、ロスアラモスのオペレータの機関動物実験委員会によって承認された実験動物とすべてのプロトコルの人道的使用のための健康のガイドラインの国立研究所を満たしていた米国エネルギー省との契約番号DE-AC52-06NA25396下の国立研究所。本研究ではマウスのケアはUCSDの動物承認されたプロトコルに準拠しています。ヒトの組織は、UCSDの承認されたIRBのプロトコルの一部として得られた。この作品は、神経疾患や脳卒中の国立研究所（ニューロ顕微鏡共用施設、UCサンディエゴ）からカリフォルニアラボ料大統領プログラム（PG）の大学とグラントNS047101から助成118645によってサポートされていました。

<ol>
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Sci. U.S.A. 105, 15064-15069 (2008).</li><li>Hooper, L. V., Macpherson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+adaptations+that+maintain+homeostasis+with+the+intestinal+microbiota.">Immune adaptations that maintain homeostasis with the intestinal microbiota.</a> Nat. Rev. Immunol. 10, 159-169 (2010).</li><li>Hayat, M. A., Hayat, 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> Microscopy, Immunohistochemistry, and Antigen Retrieval Methods: For Light and Electron Microscopy. , 53-70 (2002).</li><li>Cohen, M., Hurtado-Ziola, N., Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ABO+blood+group+glycans+modulate+sialic+acid+recognition+on+erythrocytes.">ABO blood group glycans modulate sialic acid recognition on erythrocytes.</a> Blood. 114, 3668-3676 (2009).</li><li>Cohen, M., Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+sialome--far+more+than+the+sum+of+its+parts.">The sialome--far more than the sum of its parts.</a> OMICS. 14, 455-464 (2010).</li><li>Ota, H., Katsuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternating+laminated+array+of+two+types+of+mucin+in+the+human+gastric+surface+mucous+later.">Alternating laminated array of two types of mucin in the human gastric surface mucous later.</a> Histochemical J. 24, 86-92 (1992).</li><li>Matsuo, K., Ota, H., Akamatsu, T., Sugiyama, A., Katsuyama, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemistry+of+the+surface+mucous+gel+layer+of+the+human+colon.">Histochemistry of the surface mucous gel layer of the human colon.</a> Gut. 40, 782-789 (1997).</li><li>Boyde, A., Maconnachie, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Treatment+with+lithium+salts+reduces+ethanol+dehydration+shrinkage+of+glutaraldehyde+fixed+tissue.">Treatment with lithium salts reduces ethanol dehydration shrinkage of glutaraldehyde fixed tissue.</a> Histochemistry. 66, 181-187 (1980).</li></ol>

特別な利害関係は宣言されません。

凍結組織の粘液とグリカンエピトープの保全は、パラフィン包埋した組織のものよりも優れている。我々は、分泌された粘液層の保全（ 図1および図3）、パラフィン包埋された組織と比較して凍結した組織内の3糖鎖構造（ 図3）の分布を示した。そのようなカルノア液（60％エタノール、30％クロロホルム、10％酢酸）17などの特殊な固定剤は、組織サンプル中の粘液層の最適な保全のために開発されている。理想的には、この溶液は粘液研究のために専用されており、粘液層16から17の滑らかな外観を維持することが示された組織サンプルを収集するために使用されるべきである。 10月中に埋め込まれた未定着凍結サンプルの粘液層が表示され、ゴツゴツしたいくつかの地域で組織から切り離すかもしれませんが、全体的な層の厚さはカルノア液およびembeddで固定した組織で観察されたものと一致しているパラフィン16から17のed。 16 -例えば、凍結ヒト結腸組織切片における粘液層がCarnoy&#39;s固定ヒト結腸サンプル55.4±2.5μmである（204.8μmの範囲7.7）のために報告された範囲内にある〜100μm程度（ 図1）です。 それは、何十年も前から知られており、その結果生体試料18〜30％収縮し、組織13からキシレンなどの有機溶剤、Citrisolvとある程度クロロホルム抽出脂質、糖脂質とは、タンパク質がその中のエタノール脱水の結果。固定（10％緩衝ホルマリン）、脱水（増加エタノール濃度）、およびクリア（Citrisolvまたはキシレン）：パラフィン包埋組織のための処理は、次の手順が含まれます。未固定凍結組織切片の上にこれらの手順を模倣することによって、我々はCitrisolvが（Figurパラフィン包埋組織のものに類似している組織形態が生じる凍結組織切片からの粘 ​​液を抽出することを実証e 2と、右パネル）。これとは対照的に、粘液層（ 図2、左と中央パネル）ホルマリンまたはエタノールとのインキュベーションによって変化しなかった。 Citrisolv /キシレンで長時間のインキュベーション、粘液層の崩壊の結果を必要とする標準的なパラフィン包埋手順のクリアステップ、このことが示唆している。ホルマリン固定は粘液層にダメージを与えないとホルマリンで固定した凍結組織切片を、容易にグリカン、糖脂質およびタンパク質（ 図2、図3及び図6）に対するレクチンや抗体で染色することができます。これらの効果は、膜結合タンパク質および組織病理学の研究のために無視できるかもしれないが、彼らはそのような分泌粘液層のような高度に水和した構造のために荒廃しています。しかしムチンの組織学的研究はまだ粘液層の保全が次善であるパラフィン包埋サンプルを主に行っています。そのような自体の正確なIDとして粘液層組成の深さの分析でcretedまたは膜結合MUC糖タンパク質の組み合わせは、特異的抗体とタンパク質バックボーンの同定のための質量分析を必要とします。粘液層の保全は、このような研究のための初期の要件ですが。 多くの研究室では、さまざまなプロジェクトのために、過去に収集した10月中に凍結組織サンプルを持って、これらの組織は、容易にムチン、糖脂質および粘液の保全のために独自に設計された特殊な固定液中に組織を収集する必要性を排除する糖鎖分布を調べるために使用することができます。凍結した組織では、最小限の処理を受けるため、自然の中で水和している糖鎖の天然分布は、保持されます。これは、微生物 - 宿主相互作用の分野では特に重要である。これらの &quot;障壁&quot;分子飾る分泌ムチンと多くのグリカン構造の自然主義的な分布と個体数の知識は、宿主の防御、微生物の搾取とpathogenesを理解する上で鍵となるだろうです。

<table border="1"><tbody><tr><td> 試薬や機器の名称 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> 2 - メチルブタン</td><td>フィッシャー·サイエンティフィック</td><td> 03551から4 </td></tr><tr><td> AECのペルオキシダーゼ基質キット</td><td>ベクトルラボ</td><td> SK-4200 </td></tr><tr><td>アルシアンブルー</td><td>シグマアルドリッチ</td><td> A3157 </td></tr><tr><td>抗CA19-9モノクローナル抗体</td><td>カルビオケム</td><td> CA1003 </td></tr><tr><td>抗MUC5ACモノクローナル抗体</td><td>ミリポア</td><td> MAB2011 </td></tr><tr><td>アビジン - ビオチンブロッキング·キット</td><td>ベクトルラボ</td><td> SP-2001 </td></tr><tr><td>ビオチン化ロバ抗マウス抗体</td><td>ジャクソンイムノ</td><td> 90863 </td></tr><tr><td>ビオチン化SNA </td><td>ベクトルラボ</td><td> B-1305 </td></tr><tr><td>ウシ血清albu分</td><td>シグマアルドリッチ</td><td> A4503 </td></tr><tr><td>キチン加水分解物</td><td>ベクトルラボ</td><td> SP-0090 </td></tr><tr><td>クライオスタットミクロトーム</td><td>ライカマイクロシステムズ</td><td>ライカCM 1800 </td></tr><tr><td>ヘマトキシリン</td><td> Surgipath医療ケガニ</td><td> 3801570 </td></tr><tr><td>過酸化水素30パーセント</td><td>フィッシャー·サイエンティフィック</td><td> H325-100 </td></tr><tr><td>ジャカリン - FITC </td><td>ベクトルラボ</td><td> FL-1151 </td></tr><tr><td>マイヤーのヘマトキシリン</td><td>シグマアルドリッチ</td><td> MHS32 </td></tr><tr><td>メリビオース</td><td>シグマアルドリッチ</td><td> M5500 </td></tr><tr><td>核ファーストレッド</td><td>ベクトルラボ</td><td> H-3403 </td></tr><tr><td> OCTコンパウンド</td><td> VWRインターナショナル</td><td> 25608-930 </td></tr><tr><td>ペルオキシダーゼ結合ストレプトアビジン</td><td>ジャクソンイムノ</td><td> 94638 </td></tr><tr><td>シッフ試薬</td><td>電子顕微鏡学</td><td> 26052 </td></tr><tr><td> sWGA-ローダミン</td><td>ベクトルラボ</td><td> RL1022S </td></tr><tr><td> TKH2モノクローナル抗体</td><td> ATCC </td><td> HB-9654 </td></tr><tr><td> VectaMount水性封入剤</td><td>ベクトルラボ</td><td> H-5501 </td></tr><tr><td> Cytoseal 60 </td><td>サーモサイエンティフィック</td><td> 8310から4 </td></tr><tr><td>ピールウェイ金</td><td>ポリサイエンス社</td><td> 18646A-1 </td></tr></tbody></table>

using unfixed, frozen tissues to study natural mucin distribution

ムチンは重量1-3で70％以上の炭水化物を含む複雑かつ高度にグリコシル化され、O-結合型糖タンパク質である。杯細胞と胃粘膜によって生成分泌ムチンは、腸や気道の3,4行は上皮そのマイクロメートル厚の粘液層のための足場を提供する。ムチンに加えて、粘液層は、抗菌ペプチド、サイトカイン、および免疫グロブリン5-9を含む。粘液層は、ホスト自然免疫の重要な部分であり、微生物8,10-12を侵略に対する最初の防衛線を形成している。このように、粘液は、微生物との多数の相互作用に従うものとし、両方の病原体と共生し、分泌ムチンは、これらの相互作用のための重要なインタフェースを形成します。そのような生物学的相互作用の研究は、通常、組織採取と染色のための組織学的方法を含む。組織採取とpreserv用の2つの最も一般的に使用される組織学的方法クリニックで、研究所でationは、次のとおりです。冷凍保護剤メディアに埋め込む続いパラフィン包埋し、組織の凍結が続き、ホルマリン固定。 パラフィン包埋組織サンプルは、明快さと明確に定義された形態を含む組織学的可視化のための最適な性質を持つセクションを生成します。しかし、パラフィン包埋処理中にエピトープの数が変更され、これらのエピトープを勉強するためには、組織切片は、さらに多くのエピトープ検索方法13のいずれかで処理しなければならないとなる。分泌されるムチンと脂質は有機溶剤（キシレンまたはCitrisolv）とのインキュベーションを延長する必要パラフィン包埋クリア化ステップの間に、組織から抽出されます。したがって、このアプローチでは、in vivoでのムチンや粘液の性質や分布に着目した研究のためのサブ最適です。 これとは対照的に、最適切断温度で凍結組織（OCT）は培地埋め込みボイド試料の脱水及び清算、サンプルの水和を維持します。これは、水和した粘液層のより良い保存を可能にし、従って上皮生物学におけるムチンの多数の役割の研究を可能にします。この方法は、組織の最小限の処理を必要とするため、組織は、より自然な状態で保存されています。したがって凍結組織切片は染色前に、追加の処理を必要とせず、容易に免疫組織化学法を用いて解析することができます。 我々は、冷凍結腸サンプルにおけるマイクロメートル厚の分泌粘液層の保全を実証している。同じ組織をパラフィン包埋されている場合、この層を大幅に低減されます。我々はまた、植物レクチンを用いたムチン型糖鎖上に提示されるエピトープの免疫蛍光染色を示す。このアプローチの利点は、それが特別な固定剤の使用を必要とし、すでに研究室に保存されることもあり凍結組織を活用できていないということです。

Watch this Scientific Journal Video about Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution at JoVE.com

Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

最適切断温度の媒体（10月）に埋め込まれた未固定凍結組織サンプルは分泌粘液の自然分布とグリコシル化を研究するために使用することができます。このアプローチ組織処理に最小限であり、糖脂質、ムチンおよび糖鎖エピトープの自然なプレゼンテーションが保存されています。組織切片を蛍光や発色検出を用いた免疫組織化学によって分析することができる。

天然ムチンの分布を調べ不定、凍結組織を用いた

Mucins are complex and heavily glycosylated O-linked glycoproteins, which contain more than 70% carbohydrate by weight1-3. Secreted mucins, produced by ...

using-unfixed-frozen-tissues-to-study-natural-mucin-distribution

Research

JoVE Journal

Biology

47.5K ビュー.  University of California, San Diego. 最適切断温度の媒体（10月）に埋め込まれた未固定凍結組織サンプルは分泌粘液の自然分布とグリコシル化を研究するために使用することができます。このアプローチ組織処理に最小限であり、糖脂質、ムチンおよび糖鎖エピトープの自然なプレゼンテーションが保存されています。組織切片を蛍光や発色検出を用いた免疫組織化学によって分析すること...

ビデオ: Using Unfixed, Frozen Tissues to Study Natural Mucin Distribution

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

トカゲの運動の速度と方向の視覚感度のテスト

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

生物学

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

コンピュータによって生成された動物モデルの刺激

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Using the optokinetic response to study visual function of zebrafish

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the visual functions of larval zebrafish1-5. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adult zabrafish. Here, we introduce how to measure the OKR of adult zebrafish with our simple custom-built apparatus using a new protocol which is established in our lab. Both our apparatus and step-by-step procedure of OKR in adult zebrafish are illustrated in this video. In addition, the measurements of the larval OKR, as well as the optomotor response (OMR) test of adult zebrafish, are also demonstrated in this video. This OKR assay of adult zebrafish in our experiment may last for up to 4 hours. Such OKR test applied in adult fish will benefit to visual function investigation more efficiently when the adult fish vision system is manipulated.
Su-Qi Zou and Wu Yin contributed equally to this paper.

Optokinetic response has been widely used to assess the visual functions of larval zebrafish. Nevertheless, the standard protocol for larval fish is not yet readily applicable in adults1-5. Here, we introduce how to measure the OKR of adult zebrafish using a new protocol which is established in our lab.

ゼブラフィッシュの視覚機能を研究するために視運動性応答を使用して

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

Reverse Genetic Morpholino Approach Using Cardiac Ventricular Injection to Transfect Multiple Difficult-to-target Tissues in the Zebrafish Larva

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to employ reverse genetics have not yet been adequately developed to assess gene function in regeneration or tissue homeostasis during larval stages after zebrafish embryogenesis, and several tissues within the zebrafish larva are difficult to target. Intraventricular injections of gene-specific morpholinos offer an alternative method for the current inability to genomically target zebrafish genes in a temporally controlled manner at these stages. This method allows for complete dispersion and subsequent incorporation of the morpholino into various tissues throughout the body, including structures that were formerly impossible to reach such as those in the larval caudal fin, a structure often used to noninvasively research tissue regeneration. Several genes activated during larval finfold regeneration are also present in regenerating adult vertebrate tissues, so the larva is a useful model to understand regeneration in adults. This morpholino dispersion method allows for the quick and easy identification of genes required for the regeneration of larval tissues as well as other physiological phenomena regulating tissue homeostasis after embryogenesis. Therefore, this delivery method provides a currently needed strategy for temporal control to the evaluation of gene function after embryogenesis.&#160;

An adaptable reverse genetic method for zebrafish to assess gene function during later stages of development and physiological homeostasis such as tissue regeneration using intraventricular injections of gene-specific morpholinos.

ゼブラフィッシュの幼虫の複数の困難な標的組織をトランスフェクションするために心室インジェクションを用いた遺伝子モルホリノアプローチを逆転

The zebrafish is an important model to understand the cell and molecular biology of organ and appendage regeneration. However, molecular strategies to ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

腫瘍球の高スループット画像解析：自動的かつ正確にスフェロイドのサイズを測定するための使いやすいソフトウェアアプリケーション

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...

Using Mouse Mammary Tumor Cells to Teach Core Biology Concepts: A Simple Lab Module

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in preparation for biomedical, graduate and professional programs or careers in science. To address this, a simple laboratory module was devised to teach the concepts of cell division, cellular communication and cancer through the application of animal cell culture techniques. Here the mouse mammary tumor (MMT) cell line is used to model for breast cancer. Students learn to grow and characterize these animal cells in culture and test the effects of traditional and non-traditional chemotherapy agents on cell proliferation. Specifically, students determine the optimal cell concentration for plating and growing cells, learn how to prepare and dilute drug solutions, identify the best dosage and treatment time course of the antiproliferative agents, and ascertain the rate of cell death in response to various treatments. The module employs both a standard cell counting technique using a hemocytometer and a novel cell counting method using microscopy software. The experimental procedure lends to open-ended inquiry as students can modify critical steps of the protocol, including testing homeopathic agents and over-the-counter drugs. In short, this lab module requires students to use the scientific process to apply their knowledge of the cell cycle, cellular signaling pathways, cancer and modes of treatment, all while developing an array of laboratory skills including cell culture and analysis of experimental data not routinely taught in the undergraduate classroom.

A feasible laboratory module for biology undergraduates that explores advanced cellular and molecular concepts using animal cell culture is described. Students grow, characterize and manipulate a breast cancer cell model by exposure to chemotherapy agents. Cell viability is assayed through cell counting using both a standard and novel method.

コア生物学の概念を教えるために、マウス乳腺腫瘍細胞を用いた：簡単なラボ·モジュール

Undergraduate biology students are required to learn, understand and apply a variety of cellular and molecular biology concepts and techniques in ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

腸によって脂質輸送を研究するためのCaco-2細胞を用いました

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

ムチンアガロースゲル電気泳動：高分子量の糖タンパク質のためのウェスタンブロッティング


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Deploying Community Scientists to Conduct Nondestructive Genetic Sampling of Rare Butterfly Populations

Global insect declines continue to accelerate. Effective genetic sampling is critically needed to advance the understanding of many taxa and address existing knowledge gaps. This protocol represents a demonstrated method for nondestructively sampling rare butterflies for population genetic structure or DNA barcoding analyses. It uses the chorion of hatched butterfly ovae to yield sufficiently high quantity and quality DNA for successful gene sequencing to confirm species identity and quantify genetic variation. It may be particularly useful when other tissue sampling techniques are impractical or unavailable. While developed for a lepidopteran, it nonetheless could easily be adapted for use with other insect species. It was specifically designed with ease of use as a goal to help maximize broad implementation by individuals of varying experience and skill levels, such as community scientists, conservation practitioners, and students, and for use over large geographic areas to facilitate broad population sampling. The data generated can help inform taxonomic and listing decisions, conservation and management actions, and enhance basic ecological research.

Here, we present a straightforward protocol for noninvasive genetic sampling of butterfly populations based on the field collection of residual egg debris. It can be used to confirm species identity and quantify genetic variation. This protocol can be easily adapted to broader groups for community science involvement.

希少な蝶の個体群の非破壊遺伝子サンプリングを実施するためのコミュニティ科学者の派遣

Global insect declines continue to accelerate. Effective genetic sampling is critically needed to advance the understanding of many taxa and address ...

凍結哺乳類組織からの単一核の単離とプロファイリング

A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing

Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.

著者スポットライト:創薬の強化 - 凍結組織からの核抽出のための自動化され標準化されたプロトコルの開発 

The study describes a simple, quick, and partially automated protocol to isolate high-quality nuclei from frozen mammalian tissues for downstream single nuclei RNA sequencing.