אנו מודים סרגיי Demin וטטיאנה Karamysheva על עזרו בהכנת כרומוזום ודגים על אנופלס. אנו מודים גם לדוד Severson שספקו לנו Aedes ושכפולים גנטיים DNA Culex BAC ומליסה ווייד לעריכת הטקסט. עבודה זו נתמכה על ידי שני מענקים מהמוסד הלאומי לבריאות: 1R21 AI88035-01 למריה V. Sharakhova ו1R21 AI094289-01 לאיגור V. Sharakhov.

<ol>
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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+ribosomal+RNA+gene+probe+differentiates+member+species+of+the+Anopheles+gambiae+complex.">A ribosomal RNA gene probe differentiates member species of the Anopheles gambiae complex.</a> The American journal of tropical medicine and hygiene. 37, 37-41 (1987).</li><li>Kumar, A., Rai, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosomal+localization+and+copy+number+of+18S+28S+ribosomal+RNA+genes+in+evolutionary+diverse+mosquitoes+(Diptera,+Culicidae).">Chromosomal localization and copy number of 18S+28S ribosomal RNA genes in evolutionary diverse mosquitoes (Diptera, Culicidae).</a> Hereditas. 113, 277-289 (1990).</li><li>Brown, S. E., Knudson, D. 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אין ניגודי האינטרסים הכריזו.

Nonfluorescent כלאה באתר על כרומוזומים mitotic של יתושים בוצע לראשונה בשנת 1990 על ידי א &#39;קומאר וק ראי 17. במחקר זה, 18S וגני DNA ריבוזומלי -28, משובט יחד באחד פלסמיד, הונחו לכרומוזומים של 20 מינים של יתושים. בדיקת DNA הייתה שכותרת רדיואקטיבית והכלאת הכרומוזומים מגרעיני המוח. בין שלושה סוגי יתושים, דגי טכניקה פותחה רק לכרומוזומים mitotic משורת התאים של AE. aegypti 10,18,19 ומעולם לא בוצע על כרומוזומים mitotic מיתושים חיים. לאחרונה, פתח טכניקה פשוטה וחזקה לקבלת הכנות כרומוזום באיכות גבוהה מתעודות זהות של 4 זחלים ה instar 9. שיטה זו מאפשרת למספר גבוה של הכרומוזומים שיתקבל בשקופית אחת ויכולה לשמש אוניברסלי עבור כל הזנים של יתושים. הנחיצות של שימוש sta זחל היחיד, לא גלמים או מבוגרGES של יתושים, לשקופית הכנה הוא כנראה המגבלה היחידה של השיטה. השיטה הסטנדרטית דגי 12 הייתה מותאמת לשימוש BAC שיבוט גנטי וrDNA IGS כמו בדיקות לכרומוזומים mitotic של Aedes, Culex, ואנופלס. בנוסף ליישומים הספציפיים האלה, את פרוטוקולי הדגים המתוארים כאן יכולים לשמש גם למטרות אחרות. הפרוטוקול המתקדם הדגים, אשר מנצל 0 שברי DNA t C לחסימת הכלאה נוקבת, יכול להיות מיושם גם להכלאה של שיבוטי BAC או כל מקטעי דנ&quot;א גדולים אחרים באזורי heterochromatic של אנופלס. אזורי Heterochromatic מועשרים במרכיבי transposable וחוזר אחרים, ובדיקות מהאזורים אלה בדרך כלל לייצר רקע חזק בכרומוזומים 3. שימוש 0 שברים ללא תווית C t DNA יסייע להפחית הכלאה נוקבת של החללית לכרומוזומים. הגרסה הפשוטה של ​​דגי יחסי הציבורotocol יכול לשמש לכל rDNA או בדיקות DNA חוזרים על כרומוזומים mitotic של יתושים וחרקים אחרים. בנוסף, זה יכול להיות מיושם גם להכלאה של DNA השיבוט BAC במינים בעלי תכולת DNA חוזרת נמוכה באזורי euchromatic כגון אנופלס או דרוזופילה. הפרוטוקול המוצע כאן יעזור להשיג מכלולים הגנום כרומוזום מבוססים מאוד מוגמרים, ליתושים ויכול באופן כללי לשמש ליישומי cytogenetic שונים בקבוצות אחרות של חרקים.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם המגיב / הציוד </td><td> חברה </td><td> מספר קטלוגים </td><td> תגובות </td></tr><tr><td> MZ6 stereomicroscope היקה </td><td> ליקה </td><td> VA-OM-E194-354 </td><td> Stereomicroscope שונה ניתן להשתמש </td></tr><tr><td> מיקרוסקופ השלב אולימפוס CX41 </td><td> אולימפוס </td><td> CX41 </td><td> מיקרוסקופ פאזה שונה ניתן להשתמש </td></tr><tr><td> אולימפוס BX61 מיקרוסקופ פלואורסצנטי </td><td> אולימפוס </td><td> BX61 </td><td> מיקרוסקופ פלואורסצנטי שונה ניתן להשתמש </td></tr><tr><td> Denaturation / כלת מערכת שקופיות ThermoBrite </td><td> אבוט מולקולרי </td><td> 30-144110 </td><td> משמש כבלוק חימום ותא לח </td></tr><tr><td> מחטים מבתרות </td><td> ScienceTools פיין </td><td> 10130-10</td><td></td></tr><tr><td> מחזיקי מחט </td><td> כלי מדע פיין </td><td> 26018-17 </td><td></td></tr><tr><td> מספריים מבתרים </td><td> כלי מדע פיין </td><td> 15000-03 </td><td></td></tr><tr><td> 75x25 הכפול חלבי המייקר מחליק </td><td> קורנינג </td><td> 2949-75x25 </td><td></td></tr><tr><td> coverslips מיקרוסקופ המ&quot;מ 22x22 </td><td> הפישר סיינטיפיק </td><td> 12-544-10 </td><td></td></tr><tr><td> Parafilm </td><td> הפישר סיינטיפיק </td><td> 13-374-10 </td><td></td></tr><tr><td> גומי מלט </td><td> הפישר סיינטיפיק </td><td> 50-949-105 </td><td></td></tr><tr><td> חומצה אצטית </td><td> הפישר סיינטיפיק </td><td> A491-212 </td><td></td></tr><tr><td> 200 הוכחת אלכוהול </td><td> מעבדות Decon </td><td> 2701 </td><td></td></tr><tr><td> חומצת Propionic </td><td> סיגמא Aldrich </td><td> 402907 </td><td></td></tr><tr><td> חומצת כלור </td><td> הפישר סיינטיפיק </td><td> A144-500 </td><td></td></tr><tr><td> dihydrate ציטרט הנתרן </td><td> הפישר סיינטיפיק </td><td> S279-500 </td><td></td></tr><tr><td> trihydrate יצטט נתרן </td><td> הפישר סיינטיפיק </td><td> BP334-500 </td><td></td></tr><tr><td> אשלגן כלורי </td><td> הפישר סיינטיפיק </td><td> BP366-500 </td><td></td></tr><tr><td> EDTA </td><td> הפישר סיינטיפיק </td><td> S311-500 </td><td></td></tr><tr><td> טריס בסיס </td><td> הפישר סיינטיפיק </td><td> BP152-1 </td><td></td></tr><tr><td> PBS 10x </td><td> Invitrogen </td><td> P5493 </td><td></td></tr><tr><td> 10% NBF (ניטראלי שנאגר פורמלין) </td><td> סיגמה אולדריץ </td><td> HT501128 </td><td></td></tr><tr><td> 99% פוראמיד </td><td> הפישר Scientific </td><td> BP227500 </td><td></td></tr><tr><td> מלח נתרן סולפט dextran </td><td> סיגמה אולדריץ </td><td> D8906 </td><td></td></tr><tr><td> 20x SSC חיץ </td><td> Invitrogen </td><td> AM9765 </td><td></td></tr><tr><td> 1 פתרון יודיד mM YOYO-1 (491/509) </td><td> Invitrogen </td><td> Y3601 </td><td></td></tr><tr><td> Antifade להאריך ריאגנט זהב </td><td> Invitrogen </td><td> <a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=P36930" target="_blank">P36930</a> </td><td></td></tr><tr><td> dATP, dCTP, dGTP, dTTP </td><td> Fermentas </td><td> R0141, R0151, R0161, R0171 </td><td></td></tr><tr><td> Cy3-dUTP, Cy5-dUTP </td><td> GE Healthcare </td><td> PA53022, PA55022 </td><td></td></tr><tr><td> BSA </td><td> סיגמה אולדריץ </td><td> A3294 </td><td></td></tr><tr><td> פולימראז ה-DNA אני </td><td> Fermentas </td><td> EP0041 </td><td></td></tr><tr><td> אני DNase </td><td> Fermentas </td><td> EN0521 </td><td></td></tr><tr><td> S1 nuclease </td><td> Fermentas </td><td> EN0321 </td><td></td></tr><tr><td> DNA פולימראז תקי </td><td> Invitrogen </td><td> 18038-042 </td><td></td></tr><tr><td> RNase </td><td> סיגמה אולדריץ </td><td> 9001-99-4 </td><td></td></tr><tr><td> עכלן </td><td> USB </td><td> 9001-75-6 </td><td></td></tr><tr><td> דנ&quot;א זרע סלמון </td><td> סיגמה אולדריץ </td><td> D7656 </td><td></td></tr><tr><td> Nonidet-P40 (NP40) </td><td> ביולוגי בארה&quot;ב </td><td> NC9375914 </td><td></td></tr><tr><td> דם Qiagen וMaxikit תא תרבות </td><td> Qiagen </td><td> 13362 </td><td></td></tr><tr><td> ערכת Construct גדולה Qiagen </td><td> Qiagen </td><td> 12462 </tד&gt; <td></td></tr></tbody></table>

fluorescent in situ hybridization on mitotic chromosomes of mosquitoes

פלורסנט כלאה באתר (FISH) הוא טכניקה המשמשת באופן שגרתי במעבדות רבות כדי לקבוע את המיקום הכרומוזומלי של בדיקות DNA ו-RNA. יישום החשוב של שיטה זו הוא הפיתוח של מפות באיכות גבוהה פיסיות שימושיות לשיפור המכלולים הגנום לאורגניזמים שונים. דפוס banding הטבעי של כרומוזומים polytene והמיטוטי מספק הנחיות לסידור והכיוון המדויק של supercontigs הגנומי. בין שלושה סוגי יתושי אנופלס, כלומר, Aedes, וCulex, טכניקת מיפוי כרומוזום מבוססת מבוססת היטב פותחה רק עבור אנופלס, שחבריה מחזיקים הכרומוזומים polytene קריאים 1. כתוצאה ממאמצי מיפוי הגנום, 88% מ. gambiae הגנום הושם לתפקידי כרומוזום מדויקים 2,3. שני סוגים אחרים, יתושי Aedes וCulex, שpolytenized ב הכרומוזומים גרועיםecause של ייצוג יתר משמעותי של אלמנטי transposable בגנום שלהם 4, 5, 6. רק 31 ו 9% מsupercontings הגנומי הוקצו ללא סדר או נטייה לכרומוזומים של AE. aegypti 7 וCx. quinquefasciatus 8, בהתאמה. הכנת כרומוזום mitotic לשני מינים אלה הוגבל בעבר לגרעיני מוח ושורות תאים. עם זאת, שקופיות כרומוזום שהוכנו מגרעיני המוח של יתושים בדרך כלל להכיל מספרים נמוכים של צלחות metaphase 9. כמו כן, למרות טכניקת FISH פותחה עבור כרומוזומים mitotic משורת תאים של AE. aegypti 10, ההצטברות של שחלופי כרומוזומליות מרובים בכרומוזומים 11 שורת תאים הופך אותם חסרי תועלת למיפוי הגנום. כאן אנו מתארים טכניקה פשוטה וחזקה לקבלת הכנות כרומוזום mitotic איכותיות מדיסקים דמיוניים (IDS) של 4 זחלי instar ה שגלשמש עבור כל השלושה הסוגים של יתושים. פרוטוקול סטנדרטי דגים 12 הוא מותאם לשימוש בשיבוטי BAC של הדנ&quot;א הגנומי כמו בדיקה על כרומוזומים mitotic של AE. aegypti וCx. quinquefasciatus, ולניצול spacer intergenic (IGS) אזור של דנ&quot;א ריבוזומלי (rDNA) כמו בדיקה ב. הכרומוזומים gambiae. בנוסף למיפוי פיזי, הטכניקה שפותחה ניתן ליישם ציטוגנטיקה אוכלוסייה וטקסונומיה כרומוזום / סיסטמטיקה של יתושים וקבוצות חרקים אחרות.

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Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

בין השלושה סוגי היתושים, כלומר<em&gt; אנופלס,</em&gt;<em&gt; Aedes</em&gt;, ו<em&gt; Culex</em&gt;, טכניקות מיפוי הגנום פיסיות הוקמו רק עבור<em&gt; אנופלס</em&gt;, שחבריהם בעלי כרומוזומים polytene קריאים. לסוגים של<em&gt; Aedes</em&gt; ו<em&gt; Culex</em&gt; עם זאת, מיפוי ציטוגנטית נותר מאתגר בגלל האיכות הירודה של הכרומוזומים polytene. כאן אנו מציגים פרוטוקול אוניברסלי להשגת הכנות באיכות גבוהה של כרומוזומים mitotic ופרוטוקול דגי אופטימיזציה עבור כל השלושה הסוגים של יתושים.

פלואורני<em&gt; באתר</em&gt; הכלאה על כרומוזומים mitotic של יתושים

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

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Research

JoVE Journal

Immunology and Infection

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

25.5K Views.  Virginia Tech. בין השלושה סוגי היתושים, כלומר אנופלס, Aedes, ו Culex, טכניקות מיפוי הגנום פיסיות הוקמו רק עבור אנופלס, שחבריהם בעלי כרומוזומים polytene קריאים. לסוגים של Aedes ו Culex עם זאת, מיפוי ציטוגנטית נותר מאתגר בגלל האיכות הירודה של הכר...

Video: Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

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).

שילוב של הדגימה דבק קלטת מבוססי פלואורסצנטי באתרו הכלאה עבור איתור מהיר של סלמונלה על תוצרת טרייה

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

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

נעול חומצות גרעין תזרים cytometry פלואורסצנציה באתרו</emהכלאה> (LNA זרימה FISH): שיטה לגילוי חיידקי RNA קטנים

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

הכלאה<em&gt; באתרו</em&gt; של בלוטות הרוק, השחלות, ועוברים של וקטור יתושים

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

ציור כרומוזום דו מימדי ותלות מימדי אצל יתושים מלריה

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

פלואורסצנטית כמותי באתרו היברידיזציה (דג) ו Immunofluorescence (IF) של מוצרים גנטיים ספציפיים בתאים שנדבקו KSHV

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

ויזואליזציה של קנדידה אלביקנס במערכת העיכול מוריין באמצעות פלורסנט היברידיזציה באתרו

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

וקטור יכולת ניתוחים על יתושים Aedes aegypti באמצעות וירוס זיקה

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

התפשטות הטפיל המיקרו-ספורידיאן אדהאזרדיה אדיס ב-Aedes aegypti יתושים

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

הדמיה של SARS-CoV-2 באמצעות אימונו RNA-פלואורסצנטיות בהכלאה Situ

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

הדמיה של אינטראקציות מארחות מיקרוביוטה במעיים באמצעות פלואורסצנטיות בהכלאה במקום , כתמי לטין והדמיה

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...