著者は彼らの援助が開発し、免疫組織化学を行うための技術的なミシェルのバネスの援助だけでなく、博士ペトリ-ハンソン、博士のウナギとティモシーブラウンに感謝申し上げます。このプロジェクトは、米国農務省CRISプロジェクト＃MISV - 0801310によって獣医学のミシシッピ州立大学によって資金を供給された。

<html> 1。細菌の挑戦<ol><li> 30で一晩細菌を培養する°ブレインハートインフュージョンブロスを振ってのC。 </li><li>水槽内の水の循環を停止し、（リットル当たり10ml、たとえば）100分の1の濃度で培養液を追加します。 </li><li> 1時間のインキュベーション後に、残りの細菌を洗い流すためにタンク内の水の循環を再起動します。 </li></ol> 2。サンプリング<ol><li>サンプリング時間や臓器は、研究の目的に依存しますが、我々の研究で、我々は次のサンプリングポイント使用：1、6、16、24、36、48、60、72、96、110、125および175時間後の感染を。 </li><li>鰓、側線に沿って後部の筋肉、腸、脾臓、：各時点で、水を含むMS222とサンプルに浸漬することにより、次に掲げる機関を（サンプリングされた臓器の選択は感染の過程で知られているものに基づいていた）で6魚を安楽死させる肝臓、胃、心臓、頭の腎臓、およびトランクの腎臓。 </li><li>サンプリング時には、直ちに組織カセットに臓器をロードし、1％ホルマリン、2つの時間のためにそれらを浸す。 </li><li> 2時間固定した後、ホルマリンから彼らが埋め込みされるまで残っている50％エタノールにカセットを移す。 </li><li>パラフィンは、一晩組織- Tek社VIP 3000組織のプロセッサ（さくらテック、トーランス、カリフォルニア州）とライカRM2255微分音（ライカマイクロシステムズ、バノックバーン、イリノイ州）を用いて5μm以下でセクションを使用して臓器を埋め込む。 </li></ol> 3。脱ろうセクション<ol><li>溶解するすべてのワックスを可能にするために約5分間クリア儀式で満たされた染色トラフ内のセクションを浸し。ラックを持ち上げて、余分なワックスは溶剤切る。 </li><li> 5分間クリア儀礼の第二の容器内のセクションを浸し。いくつか使用した後、第一および第二の容器の間に第一の容器と代替溶媒を交換してください。 </li><li>溶剤ワックスを除去する100％アルコールでセクションを浸し。 </li><li> 100％アルコールの第二の谷のセクションを浸し。 </li><li> 70％のアルコールの谷のセクションを浸し。 </li><li>アルコールのすべてのトレースを削除するには、水道水を流してのセクションを浸し。 </li></ol> 4。バッファの準備<ol><li>リン酸塩が7.4に蒸留水とpHを1リットルに8.0グラムのNaCl、0.2グラムのKCl、1.15グラムのNa 2 HPO 4、0.2グラムのKH 2 PO 4を溶解することにより緩衝生理食塩水の準備。 </li><li> 100mlの蒸留水にPBSの900 mlを添加することにより0.9にPBSを希釈する。 </li><li> 2ミリリットルマウス血清を添加することにより血清ブロッキング準備、200μlのトリトンX - 100と0.9 PBS 50 mlに500μlのBSA。 </li><li> 50mMの重炭酸塩緩衝生理食塩水を500 mlの蒸留H 2 Oで2.1グラムNaHCO 3と4グラムのNaClを溶解し、pHを8.2に滴定することにより調製される準備をします。 </li></ol> 5。 Immunhistochemistry <ol><li> 0.9 PBSで浸漬してセクションを再水和。 </li><li>血清をブロッキングで30分間スライドをインキュベートする。 </li><li>室温で不透明な湿室中で2時間Ed9（ブロッキング血清中で100に1希釈）でセクションをインキュベートする。 </li><li> 10分ごとにセクションをPBSで4回すすいでください。 </li><li>室温で不透明な湿室で、二次抗体を50μlで2時間のセクションを（血清ブロッキングで500倍に希釈したヤギ抗マウスFITC標識抗体）インキュベートする。 </li><li>重炭酸塩の3回緩衝生理食塩水のセクションをすすぐ。 </li><li>脱イオン水で簡単にセクションをすすいでください。 </li></ol> 6。取り付け<ol><li>数分間暗闇の中で乾燥するためにスライドしておきます。 </li><li>取付はpermafluorを使用して実行されます。 </li><li>暗い涼しい環境では、優先的に一晩、乾燥する封入剤を残す。 </li></ol> 7。顕微鏡<ol><li>組織切片は蛍光顕微鏡下で観察する準備が整いました。フィルター、私たちの実験では、我々はトリプル（MCAテキサス - レッドFITC）を装備したオリンパスBX51顕微鏡を使用していました。フォトフレームのソフトウェア（MicroFire、ゴレタ、カリフォルニア州）を用いた。 </li></ol> 8。代表的な結果： 魚組織の自家蛍光は非常に高いため、これらの組織は、便利な細菌のための背景を提供する、トライフィルタの下にオレンジ色が表示されます。 このような背景から、個々のFITC染色した細菌は、明るい緑色のドットとして表示されますと、200Xでそのロッドの形状が（図1）認識可能になります。 悪いのセクションでは、細菌の多数が一様にスライド上に存在するように見えますが、その場合には、（すすぎ段階に問題があるため）偽陽性を構成することができる。 <img src="/files/ftp_upload/2687/2687fig1.jpg" alt="図1"/> 図1二つのE.を示す筋肉切片の顕微鏡写真ictaluri（矢印aとb）（200倍）。 igure 2&quot;/&gt; 図2実験の全体スキーム

<ol>
	<li>Plumb, J. A., Woo, P. T. K., Bruno, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fish Diseases and Disorders. , 479-479 (1998).</li><li>Roberts, 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=.">.</a> Fish Pathology. , (2001).</li><li>Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Louisiana State University. , (1997).</li><li>Nusbaum, K. E., Morrisson, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+35S-Labeled+Edwardsiella+ictaluri+into+Channel+Catfish.">Entry of 35S-Labeled Edwardsiella ictaluri into Channel Catfish.</a> Journal of Aquatic Animal Health. 8, 146-146 (1996).</li><li>Karsi, A., Menanteau-Ledouble, S., Lawrence, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+bioluminescent+Edwardsiella+ictaluri+for+noninvasive+disease+monitoring.">Development of bioluminescent Edwardsiella ictaluri for noninvasive disease monitoring.</a> FEMS Microbiology Letters. 260, 286-286 (2006).</li><li>Miyazaki, T., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histopathology+of+Edwardsiella+ictaluri+in+channel+catfish,+Ictalurus+punctatus+(Rafinesque).">Histopathology of Edwardsiella ictaluri in channel catfish, Ictalurus punctatus (Rafinesque).</a> Journal of Fish Diseases. 8, 839-839 (1985).</li><li>Areechon, N., Plumb, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Edwardsiella+ictaluri+in+Channel+Catfish,+Ictalurus+punctatus.">Pathogenesis of Edwardsiella ictaluri in Channel Catfish, Ictalurus punctatus.</a> Journal of the World Mariculture Society. 14, 249-249 (1983).</li><li>Dumpala, P. R., Lawrence, M. L., Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteome+analysis+of+Edwardsiella+ictaluri.">Proteome analysis of Edwardsiella ictaluri.</a> Proteomics. 9, 1353-1353 (2009).</li><li>Thune, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signature-Tagged+Mutagenesis+of+Edwardsiella+ictaluri+Identifies+Virulence-Related+Genes,+Including+a+Salmonella+Pathogenicity+Island+2+Class+of+Type+III+Secretion+Systems.">Signature-Tagged Mutagenesis of Edwardsiella ictaluri Identifies Virulence-Related Genes, Including a Salmonella Pathogenicity Island 2 Class of Type III Secretion Systems.</a> Applied and Environmental Microbiology. 73, 7934-7934 (2007).</li><li>Karsi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+bioluminescence+mutant+screening+strategy+for+identification+of+bacterial+virulence+genes.">High-throughput bioluminescence mutant screening strategy for identification of bacterial virulence genes.</a> Applied and Environmental Microbiology. 75, 2166-2166 (2009).</li><li>Hook, E. W., Mandell, G. L., Douglas, G., Bennett, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles and Practices of Infectious Diseases. , 1700-1700 (1990).</li><li>Ainsworth, J., Capley, G., Waterstreet, P., Munson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+monoclonal+antibodies+in+the+indirect+fluorescent+antibody+technique+(IFA)+for+the+diagnosis+of+Edwardsiella+ictaluri.">Use of monoclonal antibodies in the indirect fluorescent antibody technique (IFA) for the diagnosis of Edwardsiella ictaluri.</a> Journal of Fish Diseases. 9, 439-439 (1986).</li></ol>

利害の衝突は宣言されません。

このプロトコルの詳細組織切片のナマズ病原菌エドワードシエラ属ictaluriの in situ可視化のためのテクニック。我々の知る限り、これは説明する最初のそのようなプロトコルです。 最も重要なステップは、我々の経験では、偽陽性をもたらすことができるように不十分な洗浄として本プロトコールのステップ4.4に記載の抗体の洗浄です。 <p class="jove_content"...

<table border="1"><tbody><tr><th>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td>脳インフュージョンブイヨン</td><td>ベクトンディッキンソン</td><td> 237200 </td></tr><tr><td> MS222 </td><td>アージェント研究所</td><td></td></tr><tr><td>全体のマウスの血清</td><td>カペル</td><td> 55989 </td></tr><tr><td>ヤギ抗マウスFITC </td><td>南バイオテック</td><td> 1010年から1004年</td></tr><tr><td> Permafluor </td><td>ラボのビジョン</td><td> TA - 030 - FM </td></tr><tr><td>塩化カリウム（KCl） </td><td>シグマ</td><td> P - 4504 </td></tr><tr><td>トリトン- X </td><td>シグマアルドリッチ</td><td> X100 - 6X500ML </td></tr><tr><td>ウシ血清アルブミン</td><td>シグマ</td><td> 85040C </td></tr><tr><td>リン酸カリウム（KH 2 PO 4） </td><td>シグマ</td><td> P - 5379 </td></tr><tr><td>重炭酸ナトリウム（NaHCO 3の ） </td><td>シグマ</td><td> S - 5761 </td></tr><tr><td>塩化ナトリウム（NaCal） </td><td>シグマ</td><td> S - 9625 </td></tr><tr><td>ナトリウムリン酸水素二し（Na 2 HPO 4） </td><td>シグマ</td><td> S - 9390 </td></tr></tbody></table>

use of fluorescent immuno-chemistry for the detection of edwardsiella ictaluri in channel catfish (i. punctatus) samples

エドワードシエラ属ictaluriがチャネルナマズIctalurus punctatusの主要な病原体であり、1,2年近く前に三十年を発見してきたが、これまでのところ、これらの筆者の知る限りでは、メソッドはその場可視化のために開発されていない組織切片中の細菌。 細菌の局在が4、または生物発光細菌5というラベルの放射プレートカウント3を 、使用して、以前の研究で、生体内で決定されていますが、これらの研究のほとんどは、唯一の例外は6で、総臓器レベルで行われている。この制限は、E.ので特に懸念されるictaluriは、複雑な感染サイクル1,7を有し、そしてそれは、病原性因子8,9の様々なています。 E.の複雑な相互作用そのホストとictaluriは、同じ分類の家族になっているチフス菌10日に多くの点で似ています。 ここでは、エインズワースらにより記載されたモノクローナルEd9抗体を用いた間接免疫組織化学。11を使用して細菌の検出を可能にするテクニックを説明します。 簡単に言えば、ブロッキング血清は、非特異的bidingを防ぐために、パラフィン包埋組織切片に適用されます。 E.：次に、切片を一次抗体とインキュベートされていますictaluri特異的モノクローナル抗体Ed9。過剰な抗体は、すすぎされ、FITC標識二次抗体が追加されます。洗浄後、切片を蛍光特定の封入剤でマウントされています。 E.の検出のために許容されるこのブチナマズ組織の組織切片のin situでictaluri。

Watch this Scientific Journal Video about Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples at JoVE.com

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

ここではのラベル付けを可能にする手順を説明します。<emその場で&gt;エドワードシエラ属ictaluri</em&gt;ブチナマズから組織切片で<em&gt; Ictalurus punctatus</em&gt;セカンダリとして、プライマリ、および蛍光FITC標識抗体としてモノクローナル抗体を用いた間接免疫組織化学Ed9を使用。これは、蛍光顕微鏡を用いた細菌の検出に使用できます。

の検出のための蛍光免疫化学の使用エドワードシエラ属ictaluriブチナマズで（ I. punctatus）のサンプル

While Edwardsiella ictaluri is a major pathogen of channel catfish Ictalurus punctatus and has been discovered nearly 
three decades ago 1,2, so far, to ...

use-fluorescent-immuno-chemistry-for-detection-edwardsiella-ictaluri

Research

JoVE Journal

Immunology and Infection

Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish (I. punctatus) samples

10.4K ビュー.  Mississippi State University. ここではのラベル付けを可能にする手順を説明します。エドワードシエラ属ictaluriブチナマズから組織切片で Ictalurus punctatusセカンダリとして、プライマリ、および蛍光FITC標識抗体としてモノクローナル抗体を用いた間接免疫組織化学Ed9を使用。これは、蛍光顕微鏡を用いた細菌の検出に使用できます。

ビデオ: Use of Fluorescent Immuno-Chemistry for the detection of Edwardsiella ictaluri in channel catfish I. punctatus samples

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

粘着テープベースのサンプリングと蛍光の組み合わせその場で</emの迅速検出のための>ハイブリダイゼーションサルモネラ生鮮食品について

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

免疫・感染学

Multiplex Detection of Bacteria in Complex Clinical and Environmental Samples using Oligonucleotide-coupled Fluorescent Microspheres

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated to a microbiota dominated by a number of bacterial species, including Gardnerella vaginalis, Atopobium vaginae, and others1-3. This condition is associated with a range of negative health outcomes, including HIV acquisition4, and it can be difficult to manage clinically5. Furthermore, diagnosis of BV has relied on the use of Gram stains of vaginal swab smears that are scored on various numerical criteria6,7. While this diagnostic is simple, inexpensive, and well suited to resource-limited settings, it can suffer from problems related to subjective interpretations and it does not give a detailed profile of the composition of the vaginal microbiota8. Recent deep sequencing efforts have revealed a rich, diverse vaginal microbiota with clear differences between samples taken from individuals that are diagnosed with BV compared to those individuals that are considered normal9,10, which has resulted in the identification of a number of potential targets for molecular diagnosis of BV11,12. These studies have provided a wealth of useful information, but deep sequencing is not yet practical as a diagnostic method in a clinical setting. We have recently described a method for rapidly profiling the vaginal microbiota in a multiplex format using oligonucleotide-coupled fluorescent beads with detection on a Luminex platform13. This method, like current Gram stain-based methods, is rapid and simple but adds the additional advantage of exploiting molecular knowledge arising from sequencing studies in probe design. This method therefore provides a way to profile the major microorganisms that are present in a vaginal swab that can be used to diagnose BV with high specificity and sensitivity compared to Gram stain while providing additional information on species presence and abundance in a semi-quantitative and rapid manner. This multiplex method is expandable well beyond the range of current quantitative PCR assays for particular organisms, which is currently limited to 5 or 6 different assays in a single sample14. Importantly, the method is not limited to the detection of bacteria in vaginal swabs and can be easily adapted to rapidly profile nearly any microbial community of interest. For example, we have recently begun to apply this methodology to the development of diagnostic tools for use in wastewater treatment plants.

We describe a multiplex method for the detection of microorganisms within a sample using oligonucleotide-coupled fluorescent beads. Amplicon from all organisms within a sample is hybridized to a panel of probe-coupled beads. A Luminex or Bio-Plex instrument is used to query each bead for bead type and hybridization signal.

オリゴヌクレオチド結合蛍光マイクロスフェアを使用して複雑な臨床及び環境試料中の細菌の多重検出

Bacterial vaginosis (BV) is a recurring polymicrobial syndrome that is characterized by a change in the "normal" microbiota from Lactobacillus-dominated ...

Detection of Fluorescent Nanoparticle Interactions with Primary Immune Cell Subpopulations by Flow Cytometry

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable method of analysis by flow cytometry to study nanoparticle interaction with immune cells. Primary immune cells can be easily purified from human or mouse tissues by antibody-mediated magnetic isolation. In the first instance, the different cell populations running in a flow cytometer can be distinguished by the forward-scattered light (FSC), which is proportional to cell size, and the side-scattered light (SSC), related to cell internal complexity. Furthermore, fluorescently labeled antibodies against specific cell surface receptors permit the identification of several subpopulations within the same sample. Often, all these features vary when cells are boosted by external stimuli that change their physiological and morphological state. Here, 50 nm FITC-SiO2 nanoparticles are used as a model to identify the internalization of nanostructured materials in human blood immune cells. The cell fluorescence and side-scattered light increase after incubation with nanoparticles allowed us to define time and concentration dependence of nanoparticle-cell interaction. Moreover, such protocol can be extended to investigate Rhodamine-SiO2 nanoparticle interaction with primary microglia, the central nervous system resident immune cells, isolated from mutant mice that specifically express the Green Fluorescent Protein (GFP) in the monocyte/macrophage lineage. Finally, flow cytometry data related to nanoparticle internalization into the cells have been confirmed by confocal microscopy.

Analysis of nanoparticle interaction with defined subpopulations of immune cells by flow cytometry.

フローサイトメトリーによる一次免疫細胞亜集団との蛍光ナノ粒子の相互作用を検出

Engineered nanoparticles are endowed with very promising properties for therapeutic and diagnostic purposes. This work describes a fast and reliable ...

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of immunotherapies. Here we describe the use of fluorescent target array (FTA) technology, which utilizes vital dyes such as carboxyfluorescein succinimidyl ester (CFSE), violet laser excitable dyes (CellTrace Violet: CTV) and red laser excitable dyes (Cell Proliferation Dye eFluor 670: CPD) to combinatorially label mouse lymphocytes into &#62;250 discernable fluorescent cell clusters. Cell clusters within these FTAs can be pulsed with major histocompatibility (MHC) class-I and MHC class-II binding peptides and thereby act as target cells for CD8+ and CD4+ T cells, respectively. These FTA cells remain viable and fully functional, and can therefore be administered into mice to allow assessment of CD8+ T cell-mediated killing of FTA target cells and CD4+ T cell-meditated help of FTA B cell target cells in real time in vivo by flow cytometry. Since &#62;250 target cells can be assessed at once, the technique allows the monitoring of T cell responses against several antigen epitopes at several concentrations and in multiple replicates. As such, the technique can measure T cell responses at both a quantitative (e.g.&#160;the cumulative magnitude of the response) and a qualitative (e.g.&#160;functional avidity and epitope-cross reactivity of the response) level. Herein, we describe how these FTAs are constructed and give an example of how they can be applied to assess T cell responses induced by a recombinant pox virus vaccine.

The Use of Fluorescent Target Arrays for Assessment of T Cell Responses In vivo

The ability to monitor T cell responses in detail in vivo is important for the development of our understanding of the immune response. Here we describe the use of fluorescent target arrays (FTAs) in an in vivo T cell assay that assesses &#62;250 parameters simultaneously by flow cytometry.

T細胞応答の評価のための蛍光ターゲット配列の使用<em&gt;インビボ</em

The ability to monitor T cell responses in vivo is important for the development of our understanding of the immune response and the design of ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

の検出のための磁気スターラー方法<em&gt;旋毛虫</em筋肉試料中&gt;幼虫

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

迅速・安全・簡易マニュアル ベッドサイド核酸抽出の全血試料中のウイルス検出

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

サルモネラ属菌のスクリーニングのための高スループット プラットフォーム/赤痢菌属

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Multiplexed Fluorescent Immunohistochemical Staining, Imaging, and Analysis in Histological Samples of Lymphoma

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic purposes. However, the visualization and quantification of multiple antigens in a single tissue section using conventional chromogenic IHC is challenging. Multiplexed imaging is especially relevant in lymphoma research and diagnostics, where markers have to be interpreted in the context of a complex tumor microenvironment. Here we describe a protocol for multiplexed fluorescent IHC staining to enable the quantitative assessment of multiple targets in specific cell types of interest in lymphoma.The method covers aspects of antibody validation, antibody optimization, the multiplex optimization with markers of lymphoma subtypes, the staining of tissue microarray (TMA) slides, and the scanning of the slides, followed by data analysis, with specific reference to lymphoma. Using this method, scores for both the mean intensity of a marker of interest and the percentage positivity are generated to facilitate further quantitative analysis. Multiplexing minimizes sample utilization and provides spatial information for each marker of interest.

Here we describe a protocol for multiplex fluorescent immunohistochemical staining and imaging for the simultaneous localization of multiple cancer-associated antigens in lymphoma. This protocol can be extended to the colocalization analysis of biomarkers within all tissue sections.

多重蛍光免疫組織化学染色、イメージング、およびリンパ腫の組織学的試料中の分析

Immunohistochemical (IHC) methods for the in-situ analysis of protein expression by light microscopy are a powerful tool for both research and diagnostic ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

水サンプル中のウイルス検出のための利用ポイント方法VirWaTest

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food ...

Visualization of Bacterial Resistance using Fluorescent Antibiotic Probes

Fluorescent antibiotics are multipurpose research tools that are readily used for the study of antimicrobial resistance, due to their significant advantage over other methods. To prepare these probes, azide derivatives of antibiotics are synthesized, then coupled with alkyne-fluorophores using azide-alkyne dipolar cycloaddition by click chemistry. Following purification, the antibiotic activity of the fluorescent antibiotic is tested by minimum inhibitory concentration assessment. In order to study bacterial accumulation, either spectrophotometry or flow cytometry may be used, allowing for much simpler analysis than methods relying on radioactive antibiotic derivatives. Furthermore, confocal microscopy can be used to examine localization within the bacteria, affording valuable information about mode of action and changes that occur in resistant species. The use of fluorescent antibiotic probes in the study of antimicrobial resistance is a powerful method with much potential for future expansion.

Fluorescently tagged antibiotics are powerful tools that can be used to study multiple aspects of antimicrobial resistance. This article describes the preparation of fluorescently tagged antibiotics and their application to studying antibiotic resistance in bacteria. Probes can be used to study mechanisms of bacterial resistance (e.g., efflux) by spectrophotometry, flow cytometry, and microscopy.