저자들은 이후 적응과에서 하이브 리다이 제이션을 위해 여기에서 설명한 프로토콜을 개발하기 위해 수정된 아노 펠 레스 gambiae의 배아의 현장에서 하이브 리다이 제이션을위한 프로토콜의 논의 부드러운 조직 및 유리 제품 Goltsev위한 원위치 방법으로 개발 하이브 리다이 제이션의 조언 마리카 월터스 감사드립니다 Aedes과 Culex의 배아의 현장. 또한 아담 정지와 데이빗 Kosman 준 유용한 권장 사항을 인정합니다. 우리는 과학적 논의 Osvaldo Marinotti 감사 및 프로토콜 텍스트 편집.

<ol>
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저자는 전혀 공개가 없습니다.

현장과 colorimetric 염색법 프로토콜의 하이브 리다이 제이션은 전체 마운트 모기 조직 및 배아를 보시려면 여기를 제시하는 것은 특정 장기 및 세포 유형 내에서 성적의 지방화를위한 유용한 기술입니다. 이러한 절차는 우리 이전에 보고된 방법을 통해 모두 광범위한 세차 단계를 능률화 및 추가 기술적인 세부 사항 및 시약 소스를 제공하는 개선입니다. 우리의 경험에서 하이브 리다이 제이션 신호 colorimetric 감지 형광 기반의 탐지 방식에 비해 하이브리드화 신호의 감도와 선명도에서 우수합니다. 또한 colorimetric 감지 본질적 자동 형광 있습니다 배아에서 신호 차별과 관련된 문제를 circumvents. 낮은 풍부 성적이 타겟으로하는 경우 하이브리드화 신호 검출에 한계가 발생 배경 염색법은 분명하다. 65 하이브리드화 온도를 높이면 ° C는 다시 감소하는 것으로 확인되었습니다지상 하이브리드화 신호, 독특 대상 특정 RNA 프로브를 설계를위한 제안 대체할 수 없습니다. 이 프로토콜은 전체 마운트 타액 Aedes aegypti의 분비와 난소 및 아노 펠 레스 gambiae, 아노 펠 레스 stephensi, 애의 배아의 현장에서 하이브 리다이 제이션을 수행하는 데 사용되었습니다. aegypti 및 Culex quinquefasciatus. 이 방법은 또한 다른 모기 조직에 적용, 그리고 아마도 다른 곤충 분들입니다. 또한, 세 벡터 모기, 애의 배아 발달의 준비에 대해 처음으로 비교 지침에 대해 컴파일된. aegypti,. gambiae 및 CX. fatigans. 관찰은 신중하고 양육 조건이 세 종류의 특정 변종을 위해보고되었습니다. 그것은 발달 시간 코스 모기 종의 서로 다른 변종과 다른 양육 조건 하에서 다를 수 있습니다하는 것이 중요합니다. 현장 보충제의 Hybridizations 아나에 노력을에 지속적모기와 다른 arthropods의 transcriptomes을 lyze, 이러한 생물의 유전자 발현의 조절의 더 나은 사진을 제공할 수 있습니다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> 0.5 M EDTA (에틸렌 다이아 민 테트라 초산) </td><td> Ambion </td><td> AM9261 </td><td></td></tr><tr><td> 1M 트리스-HCL </td><td> Ambion </td><td> AM9855G </td><td> pH8.0 </td></tr><tr><td> 10X PBS </td><td> Ambion </td><td> AM9625 </td><td></td></tr><tr><td> 20X SSC </td><td> Ambion </td><td> AM9763 </td><td></td></tr><tr><td> 1.5 ML의 microfuge 튜브 </td><td> Ambion </td><td> AM12400 </td><td> 샘플 영상에서 에이즈, 이하 표준 튜브보다 불투명 </td></tr><tr><td> Deionized 포름 아미드 </td><td> Ambion </td><td> AM9342 </td><td> 4에 보관 ° C </td></tr><tr><td> DEPC 물 </td><td> Ambion </td><td> AM9932 </td><td></td></tr><tr><td> Proteinase K </td><td> Ambion </td><td> AM2546 </td><td></td></tr><tr><td> 5.25 %의 나트륨 차아 염소 산염 </td><td> 오스틴의 </td><td></td><td> A-1 브랜드 </td></tr><tr><td> T3 RNA 중합 효소 플러스 </td><td> Ambion </td><td> AM2733 </td><td> -20 ° C에서 보관 </td></tr><tr><td> T7 RNA 중합 효소의 </td><td> Ambion </td><td> AM2082 </td><td> -20 ° C에서 보관 </td></tr><tr><td> 95 % 에탄올 </td><td> 피셔 과학 </td><td> AC61511-0040 </td><td></td></tr><tr><td> Fisherbrand 일회용 폴리에틸렌 전송 pipettes </td><td> 피셔 과학 </td><td> 13-711-7M </td><td></td></tr><tr><td> 37%의 포름 알데히드 </td><td> 피셔 과학 </td><td> F79-500 </td><td></td></tr><tr><td> HPLC 등급의 메탄올 </td><td> 피셔 과학 </td><td> A452-1 </td><td></td></tr><tr><td> 마그네슘 염화물 </td><td> 피셔 과학 </td><td> M87-500 </td><td></td></tr><tr><td> 현미경 덮개 유리 </td><td> 피셔 과학 </td><td> 12-542A </td><td> 18 x18 mm </td></tr><tr><td> N-헵탄 </td><td> 피셔 과학 </td><td> H350-1 </td><td></td></tr><tr><td> P-크실렌 </td><td> 피셔 과학 </td><td> O5082-500 </td><td></td></tr><tr><td> Pyrex 9 잘 스팟 플레이트 </td><td> 피셔 과학 </td><td> 13-748B </td><td> 100x85 mm </td></tr><tr><td> 염화나트륨 (소금) </td><td> 피셔 과학 </td><td> AC32730-0025 </td><td></td></tr><tr><td> 수산화 나트륨 </td><td> 피셔 과학 </td><td> SS255-1 </td><td></td></tr><tr><td> Superfrost / 플러스 현미경 슬라이드 </td><td> 피셔 과학 </td><td> 12-550-15 </td><td> 25x75x1.0 mm </td></tr><tr><td> Davlyn 레드 클리어 - 라이너 가발 테이프 </td><td> 직접 헤어 </td><td> RED-75R12 </td><td> 폴리 / X 12야드 테이프 롤의 피부 기지 재료 0.75 </td></tr><tr><td> 하나와 Sequening위한 TOPOTA 클로닝 키트 Top10 화학적-관할 E를 샷 대장균 </td><td> Invitrogen </td><td> K457501 K457540 </td><td> 20 반응 40 반응 </td></tr><tr><td> Sonicated 연어 정자 DNA </td><td> Invitrogen </td><td> 15632-011 </td><td> -20 ° C에서 보관 </td></tr><tr><td> 안티 digoxigenin-AP 팹 조각 </td><td> 로슈 응용 과학 </td><td> 1,093,274 </td><td> 4에 보관 ° C </td></tr><tr><td> 쫄지마 RNA 라벨 믹스 </td><td> 로슈 응용 과학 </td><td> 1,277,073 </td><td> -20 ° C에서 보관 </td></tr><tr><td> NBT / BCIP 재고 솔루션 </td><td> 로슈 응용 과학 </td><td> 1,681,451 </td><td> 4에 보관 ° C </td></tr><tr><td> 서양 차단 시약 </td><td> 로슈 응용 과학 </td><td> 11921673001 </td><td> 4에 보관 ° C </td></tr><tr><td> Saatilene Hitech 폴리에스터 메쉬 (330.130) </td><td> Saati 인쇄 </td><td> 330.34 UO PW </td><td> 330 스레드 / 인치, 34 마이크론 나사 직경, 오렌지 색상 </td></tr><tr><td> 글리세린 </td><td> 시그마 </td><td> G6279-1 </td><td> PBT 70 % </td></tr><tr><td> 헤파린 나트륨 소금 </td><td> 시그마 </td><td> H3393 </td><td></td></tr><tr><td> 십대 초반 20 </td><td> 시그마 </td><td> P1379-500 </td><td></td></tr><tr><td> 37%의 포름 알데히드 </td><td> 테드 펠라 </td><td> 18,508 </td><td> 호박 ampoules 10 ML aliquots </td></tr><tr><td> 16온스 뚜껑있는 솔로 종이 용기 </td><td> 제지 회사 </td><td> SOLOKH16AJ8000 </td><td></td></tr><tr><td> Borosilicate 유리 scintillat무소속의 스크루 캡과 이온 유리병 </td><td> VWR 인터내셔널 </td><td> 66022-128 </td><td> 500 20 ML 케이스 </td></tr><tr><td> 봉인된 에어 버블 랩 celluar 쿠션 재료 </td><td> VWR 인터내셔널 </td><td> 500018-081 </td><td> 10 피트 / 롤 0.188 인치 두께 </td></tr><tr><td> 닭 혈청 </td><td></td><td></td><td> 전체 혈액 중 날개 정맥이나 소년 닭의 심장 주사에 의해 수집되었다. 혈액은 coagulated 후 30 분 동안 얼음에 배치 때까지 1 H에 대해 37 ° C에서 incubated되었다. 혈청 10 분 3,000 XG에서 수집된 및 centrifuged되었다. 결과 뜨는가 (혈청을 명확히) 사용하기 전까지 수집 및 -20 ° C에 보관되었다. </td></tr></tbody></table> 표 4. 현장에서 하이브 리다이 제이션을위한 특정 시약 및 장비의 표.

hybridization in situ of salivary glands, ovaries, and embryos of vector mosquitoes

모기 arboviruses, protozoan 기생충 및 nematodes 포함한 병원균의 다양한 집합에 대한 벡터입니다. 재생에 관련된 모기 호스트와 병원체 상호 작용, 그리고 장기에이 모기 질병일 전송을 줄이고 달걀 개발을 중단하는 전략에 대해 큰 관심되는 티슈로 표현되는 성적 증명서 및 유전자 규제 당국의 조사. 도구의 숫자는 유전자 발현의 시간적 및 조직 관련 규정을 연구하고 검증 고용되었습니다. 여기서는 특정 유전자가 표현하고 그들의 제품이 축적되는 곳이 우리의 이해를 향상시켜 공간적 정보를 얻기 위해 개발되었습니다 프로토콜을 설명합니다. 설명한 프로토콜은 다시하는 표현을 확인하고 이러한 여성 침샘뿐만 아니라 난소와 배아의 subcellular 구획 등 모기 매개로 병원체 전송에 관련된 조직에 성적 축적 패턴을 결정하는 데 사용되었습니다모기 복제 및 개발에 늦었어. 다음 절차는 대상 특정 하이브리드화 신호의 손실없이 프로토콜의 여러 단계의 효율성을 향상 최적화된 방법론을 나타냅니다. 수집, 고정, 사전 하이브리드화 및 모기 배아의 하이브 리다이 제이션을위한 구체적인 단계는 부품 B.에 상세히하는 동안 RNA 프로브 준비에 대한 지침은 부드러운 조직 및 고정과 하이브 리다이 제이션을위한 일반적인 절차의 해부는 부분에 설명되어 있습니다

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

시간적 및 공간적 유전자 발현 분석은 기능 유전체학에 중요한 역할을합니다. 전체 마운트 하이브리드화<em&gt; 현장에서</em&gt; 조직 및 subcellular 구획 내에서 성적의 지방화를 결정하는 데 유용합니다. 여기 하이브 리다이 제이션을 설명<em&gt; 현장에서</em모기의 특정 대상 조직에 대한 수정&gt; 프로토콜.

이종 교잡<em&gt; 현장에서</em&gt; 침샘, 난소, 그리고 벡터 모기의 태아의

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

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Immunology and Infection

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

16.4K 조회수.  University of California, Irvine. 시간적 및 공간적 유전자 발현 분석은 기능 유전체학에 중요한 역할을합니다. 전체 마운트 하이브리드화 현장에서 조직 및 subcellular 구획 내에서 성적의 지방화를 결정하는 데 유용합니다. 여기 하이브 리다이 제이션을 설명 현장에서 프로토콜.

비디오: Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector 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).

접착제 테이프 기반의 샘플링 및 형광의 조합 현장에서</em의 신속한 감지를 위해> 하이브 리다이 제이션 살모넬라균 신선한 농산물에 대한

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

연구

면역학 및 감염병학

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

베로 세포 배양 분석하여 필드 수집 모기에서 감염성 바이러스의 검출

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne relapsing fever (Borrelia spp.), Rocky Mountain Spotted fever (Rickettsia rickettsii), ehrlichiosis (Ehrlichia chaffeensis and E. equi), anaplasmosis (Anaplasma phagocytophilum), encephalitis (tick-borne encephalitis virus), babesiosis (Babesia spp.), Colorado tick fever (Coltivirus), and tularemia (Francisella tularensis) 1-8. To be properly transmitted into the host these infectious agents differentially regulate gene expression, interact with tick proteins, and migrate through the tick 3,9-13. For example, the Lyme disease agent, Borrelia burgdorferi, adapts through differential gene expression to the feast and famine stages of the tick's enzootic cycle 14,15. Furthermore, as an Ixodes tick consumes a bloodmeal Borrelia replicate and migrate from the midgut into the hemocoel, where they travel to the salivary glands and are transmitted into the host with the expelled saliva 9,16-19.
As a tick feeds the host typically responds with a strong hemostatic and innate immune response 11,13,20-22. Despite these host responses, I. scapularis can feed for several days because tick saliva contains proteins that are immunomodulatory, lytic agents, anticoagulants, and fibrinolysins to aid the tick feeding 3,11,20,21,23. The immunomodulatory activities possessed by tick saliva or salivary gland extract (SGE) facilitate transmission, proliferation, and dissemination of numerous tick-borne pathogens 3,20,24-27. To further understand how tick-borne infectious agents cause disease it is essential to dissect actively feeding ticks and collect tick saliva. This video protocol demonstrates dissection techniques for the collection of hemolymph and the removal of salivary glands from actively feeding I. scapularis nymphs after 48 and 72 hours post mouse placement. We also demonstrate saliva collection from an adult female I. scapularis tick.

Saliva, Salivary Gland, and Hemolymph Collection from Ixodes scapularis Ticks

The collection of infected tick hemolymph, salivary glands, and saliva is important to study how tick-borne pathogens cause disease. In this protocol we demonstrate how to collect hemolymph and salivary glands from feeding Ixodes scapularis nymphs. We also demonstrate saliva collection from female I. scapularis adults.

Ixodes Scapularis 진드기의 타액, 침샘 및 Hemolymph 컬렉션

Ticks are found worldwide and afflict humans with many tick-borne illnesses. Ticks are vectors for pathogens that cause Lyme disease and tick-borne ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

형광성의<em&gt; 현장에서</em모기의 Mitotic의 염색체에&gt; 하이브리드

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

A Method for the Measurement of Salivary Gland Function in Mice

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced saliva production. Similarly, saliva production is severely compromised in patients receiving radiation treatment for head and neck cancers. Rodent models, developed to mimic these clinical conditions, facilitate an understanding of the disease pathogenesis and allow for the development of new therapeutic strategies. Therefore, the ability to accurately, reproducibly, and repeatedly measure salivary gland function in animal models is critical. Building on procedures previously described in the literature, a method was developed that meets these criteria and was used to evaluate salivary gland function in mice. An additional advantage of this new method is that it is easily mastered, and has little inter-operator variation. Salivary gland function is evaluated as the amount (weight or volume) or rate (mL/min) of saliva produced in response to pilocarpine stimulation. The collected saliva is a good source for the analyses of protein content, immunoglobulin concentrations, and other biomolecules.

Salivary gland hypofunction is a frequent consequence of autoimmune disease and radiation therapy. Reproducible evaluation of salivary gland function in mouse models of these diseases is a technical challenge. Here, a simple method for accurate and reproducible measurement of saliva production in mice is described.

쥐에 있는 침 샘 기능의 측정 하는 방법

Patients with Sj&#246;gren&#39;s syndrome, an autoimmune disease affecting the exocrine glands, develop salivary gland inflammation and have reduced ...

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).

KSHV 감염 세포에서 특정 유전자 제품의 시투 혼성화 (FISH) 및 면역 형광 (IF)의 정량형 형광

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

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

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.

시투 혼성화에서 형광을 사용하여 뮤린 위장에서 칸디다 알비칸스의 시각화

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.

지카 바이러스를 사용하여 Aedes aegypti 모기에 벡터 역량 분석

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

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.

시투 혼성화에서 면역 RNA-형광을 이용한 SARS-CoV-2의 시각화

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