Gli autori desiderano riconoscere l&#39;assistenza tecnica di Banes Michelle e Petrie-Hanson, Eels e il Dott. Timothy Brown, per la loro assistenza sviluppare ed eseguire il immunoistochimica. Questo progetto è stato finanziato dalla USDA CRIS progetto # MISV-0801310 e dal Mississippi State University College di Medicina Veterinaria.

<html> 1. Sfida batterica <ol><li> Crescere i batteri notte a 30 ° C in agitazione Cuore brodo cervello Infusion. </li><li> Interrompere la circolazione dell&#39;acqua nelle vasche di pesce e aggiungere il brodo nella concentrazione di 1 a 100 (10 ml per litro, per esempio). </li><li> Dopo un ore di incubazione, riavviare la circolazione dell&#39;acqua nelle vasche per scovare i batteri rimasti. </li></ol> 2. Campionatura <ol><li> Tempi di campionamento e gli organi dipende l&#39;obiettivo dello studio, ma nel nostro studio, abbiamo utilizzato i seguenti punti di campionamento: 1, 6, 16, 24, 36, 48, 60, 72, 96, 110, 125 e 175 ore dopo l&#39;infezione . </li><li> Ad ogni punto del tempo, eutanasia sei pesci per immersione in acqua contenente MS222 e degustare i seguenti organi (selezione degli organi campionato era basata su ciò che è noto il processo di infezione): branchie, muscoli posteriori lungo la linea laterale, intestino, milza, fegato, stomaco, cuore, rene testa, tronco e del rene. </li><li> Su di campionamento, immediatamente carico degli organi in cassette istologiche e immergerli per due ore in 1% formalina. </li><li> Dopo due ore di fissaggio, trasferire le cassette da formalina al 50% di etanolo, dove rimangono fino incorporamento. </li><li> Paraffina incorporare organi durante la notte con un tessuto-Tek VIP 3000 processore tessuto (Sakura Tek, Torrance, California) e la sezione a 5 micron con una Leica RM2255 microtone (Leica Microsystems, Bannockburn, Illinois). </li></ol> 3. Sezioni deceratura <ol><li> Immergere le sezioni in una vasca piena di colorazione chiara rito per circa 5 minuti per consentire a tutti di sciogliere la cera. Sollevare il rack e drenare l&#39;eccesso di cera solvente. </li><li> Immergere le sezioni in un secondo contenitore di chiaro-rito per 5 minuti. Dopo alcuni usi, sostituiscono il solvente nel contenitore prima e si alternano tra i contenitori prima e la seconda. </li><li> Immergere le sezioni in 100% di alcol per rimuovere la cera solvente. </li><li> Immergere le sezioni in una depressione seconda alcol al 100%. </li><li> Immergere le sezioni in una depressione del 70% di alcol. </li><li> Immergere le sezioni in acqua corrente per rimuovere ogni traccia di alcol. </li></ol> 4. Tampone di preparazione <ol><li> Preparare tampone fosfato sciogliendo 8,0 g NaCl, 0,2 g di KCl, 1,15 g di Na 2 HPO 4 e 0,2 g di KH 2 PO 4 in 1 litro di acqua distillata e pH a 7,4. </li><li> Diluire PBS a 0.9x con l&#39;aggiunta di 900 ml di PBS a 100 ml di acqua distillata. </li><li> Preparare il blocco del siero con l&#39;aggiunta di 2 ml di siero di topo; 200 ul Triton X-100 e 500 BSA microlitri a 50 ml di PBS 0.9x. </li><li> Preparare 50 mM Buffered Saline bicarbonato si prepara sciogliendo 2,1 g NaHCO 3 e 4 g di NaCl in 500 ml di H 2 O distillata e titolato a pH 8.2. </li></ol> 5. Immunhistochemistry <ol><li> Reidratare sezioni per immersione in PBS 0.9x. </li><li> Incubare i vetrini per 30 minuti in blocco siero. </li><li> Incubare le sezioni in Ed9 (diluito 1 su 100 nel siero di blocco) per due ore in una camera opaco umida a temperatura ambiente. </li><li> Risciacquare sezioni 4 volte in PBS per 10 minuti ciascuno. </li><li> Incubare sezioni per due ore in 50 l di anticorpo secondario (anticorpi di capra anti topo marcato con FITC; diluito 500 volte a bloccare siero) in una camera opaco umida a temperatura ambiente. </li><li> Sciacquare le sezioni 3 volte in soluzione salina tamponata con bicarbonato. </li><li> Sciacquare sezioni brevemente in acqua deionizzata. </li></ol> 6. Montaggio <ol><li> Lasciare i vetrini ad asciugare al buio per alcuni minuti. </li><li> Montaggio viene eseguita utilizzando permafluor. </li><li> Lasciare il mezzo di montaggio a secco, preferibilmente durante la notte, in un ambiente buio fresco. </li></ol> 7. Microscopio <ol><li> Sezioni istologiche sono pronti ad osservare al microscopio a fluorescenza. Nel nostro esperimento, abbiamo usato una Olympus BX51 microscopio equipaggiato con una tripla (CIC; MCA Texas-Red) filtro. Il software Picture Frame (MicroFire, Goleta, California) è stato utilizzato. </li></ol> 8. Rappresentante dei risultati: Poiché l&#39;auto-fluorescenza dei tessuti dei pesci è così alta, questi tessuti apparirà arancione sotto il Tri-filtro, comodamente fornendo uno sfondo per i batteri. In questo contesto, l&#39;individuo batteri FITC macchiato apparirà come puntini luminosi verdi e, a 200X la loro forma asta sarà riconoscibile (Fig. 1). Sezioni Bad può costituire falsi positivi (a causa di un problema nella fase di risciacquo), nel qual caso un gran numero di batteri sembra essere presente uniformemente sulla slide. <img src="/files/ftp_upload/2687/2687fig1.jpg" alt="Figura 1"/> Figura 1. Micrografia di un muscolo che mostra due sezioni E. ictaluri (frecce a e b) (200x). IGURA 2 &quot;/&gt; Figura 2. Schema generale della sperimentazione

<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>

Nessun conflitto di interessi dichiarati.

Questo protocollo dettagli di una tecnica per la visualizzazione in situ del patogeno pesce gatto Edwardsiella ictaluri in sezioni istologiche. Per quanto a nostra conoscenza, questo è il primo protocollo come descritto. La fase più critica, nella nostra esperienza, è il risciacquo degli anticorpi, come descritto nel passaggio 4.4 del presente protocollo come il lavaggio insufficiente può portare a falsi positivi. Il problema principale con ...

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Cervello infusione brodo </td><td> Becton Dickinson </td><td> 237200 </td></tr><tr><td> MS222 </td><td> Argent Labs </td><td></td></tr><tr><td> Topo siero intero </td><td> Cappel </td><td> 55989 </td></tr><tr><td> Capra anti-mouse FITC </td><td> Southern Biotech </td><td> 1010-04 </td></tr><tr><td> Permafluor </td><td> Vision Lab </td><td> Ta-030-FM </td></tr><tr><td> Cloruro di potassio (KCl) </td><td> Sigma </td><td> P-4504 </td></tr><tr><td> Triton-X </td><td> Sigma-Aldrich </td><td> X100-6X500ML </td></tr><tr><td> Albumina di siero bovino </td><td> Sigma </td><td> 85040C </td></tr><tr><td> Fosfato di potassio (KH 2 PO 4) </td><td> Sigma </td><td> P-5379 </td></tr><tr><td> Bicarbonato di sodio (NaHCO 3) </td><td> Sigma </td><td> S-5761 </td></tr><tr><td> Cloruro di sodio (NaCal) </td><td> Sigma </td><td> S-9625 </td></tr><tr><td> Fosfato di sodio bibasico (Na 2 HPO 4) </td><td> Sigma </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

Mentre Edwardsiella ictaluri è un patogeno importante canale di pesce gatto Ictalurus punctatus ed è stato scoperto quasi tre decenni fa 1,2, finora, per quanto a conoscenza di questi autori &#39;, nessun metodo è stato sviluppato per consentire la visualizzazione in situ della batteri in sezioni istologiche. Mentre la localizzazione batterica è stata determinata in vivo negli studi precedenti con conta piastra 3, radiometriche etichettati 4 o batteri bioluminescenti 5, la maggior parte di questi studi sono stati condotti solo a livello di organo lordo, con una sola eccezione 6. Questa limitazione è particolarmente preoccupante perché E. ictaluri ha un ciclo complesso infezione 1,7, ed ha una varietà di fattori di virulenza 8,9. La complessa interazione di E. ictaluri con il suo ospite è simile per molti aspetti a Salmonella typhi 10, che si trova nella stessa famiglia tassonomica. Qui si descrive una tecnica che permette per la rilevazione di batteri utilizzando indiretto immunoistochimico utilizzando l&#39;anticorpo monoclonale Ed9 descritto da Ainsworth et al. 11. In breve, un siero blocco viene applicato paraffina sezioni istologiche incorporato per prevenire non specifici aspettando. Poi, le sezioni vengono incubate con l&#39;anticorpo primario: E. ictaluri anticorpo monoclonale specifico Ed9. Anticorpi in eccesso vengono risciacquati via e la FITC anticorpi marcati secondari vengono aggiunti. Dopo il risciacquo, le sezioni sono montate con una fluorescente medio di montaggio specifiche. Questo ha permesso per la rilevazione di E. ictaluri in situ in sezioni istologiche di tessuti pesce gatto di canale.

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

Qui si descrive una procedura che consenta l&#39;etichettatura dei<em&gt; Edwardsiella ictaluri in situ</em&gt; In sezioni istologiche di pesce gatto di canale<em&gt; Ictalurus punctatus</em&gt; Utilizzando l&#39;immunoistochimica indiretta con anticorpi monoclonali Ed9 come anticorpi FITC primario, e fluorescenti etichettato come secondario. Questo ha permesso per la rilevazione del batterio usando la microscopia a fluorescenza.

L&#39;uso di Immuno-fluorescenza Chimica per il rilevamento di Edwardsiella ictaluri In pesce gatto di canale ( I. punctatus) I campioni

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 Views.  Mississippi State University. Qui si descrive una procedura che consenta l'etichettatura dei Edwardsiella ictaluri in situ In sezioni istologiche di pesce gatto di canale Ictalurus punctatus Utilizzando l'immunoistochimica indiretta con anticorpi monoclonali Ed9 come anticorpi FITC primario, e fluorescenti etichettato come secondario. Questo ha permesso per la rilevazione del batterio usando la microscopia a fluorescenza.

Video: 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).

Combinazione di nastro adesivo a base di campionamento e di fluorescenza In situ</emIbridazione> per il rilevamento rapido di Salmonella Su prodotti freschi

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

Ricerca

Immunologia e infezioni

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.

Rilevazione multiplex di batteri in campioni clinici complessi ed Ambientali con oligonucleotidi accoppiati microsfere fluorescenti

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.

Rilevamento di nanoparticelle fluorescenti Interazioni con Primaria immunitario sottopopolazioni cellulari mediante citometria di flusso

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.

L&#39;uso di fluorescenti target matrici per la valutazione delle cellule T risposte<em&gt; In vivo</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.

Metodo dell&#39;agitatore magnetico per la rilevazione di<em&gt; Trichinella</em&gt; Le larve in campioni di muscolo

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).

Rapido, sicuro e semplice manuale sul comodino estrazione dell'acido nucleico per la rilevazione del Virus in campioni di sangue intero

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.

Una piattaforma di High-throughput per lo Screening della 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 ...

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.

La macchiatura di Immunohistochemical fluorescente multiplex, Imaging e analisi in campioni istologici di linfoma

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.

VirWaTest, un metodo point-of-use per il rilevamento di virus nei campioni d'acqua

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.