이 작품은 NIAID에서 NRM 및 JLB에 SREB의 논문의 화목 상을 부여하는 번호 R01AI055001 및 R01AI082127 지원했다.

1. 플루오레신는 dextran과 GSL I - isolectin B 네 얼굴 정맥 주사가 배아 vasculature를 라벨에 표시 <ol><li> isolectin B 1.5mL Eppendorf 튜브에 PBS에서 4 (20ug/200uL) 37에 따뜻한 ° C. - 인산에 FITC - dextran (50ug/mL)를 준비 식염수 (PBS) 또는 GSL 1 버퍼 그린 / PBS GSL 1 FITC - dextran 솔루션 (총 볼륨 1mL) 및 주식 1.25mM 빨리 180uL 주식 1.25mM 빠른 그린 / PBS의 100uL 추가 - isolectin 솔루션은되도록 B 4 (총 볼륨 200uL) 눈에 파란색. </li><li> allantoic 스토킹 (제대 동맥과 정맥)은 그대로두고, 함께 E14.5 - E18.5 배아와 난황을 해부하다. </li><li> 새로운 petrie 요리 (60 X 15mm)로 배아를 전송하고 실온에서 PBS에 젖어. </li><li> 머리 / 얼굴의 화살보기를 제공하기 위해 배아를 놓습니다. 부드럽게 머리에있는 배아를 파악하기 위해 마이크로 해부 집게를 사용하십시오. </li><li> 30G 바늘을 사용 50uL FITC - dextran (50ug/mL) 또는 GSL 1 삽입 - isolectin B 4 (200uL PBS에서 20ug)를 머리 뒤쪽 방향으로 바늘을 가리키는 얼굴 정맥에. </li><li> 염료가 제대 정맥에서 볼 때, 바늘을 제거하고 all​​antoic 스토킹 (제대 동맥과 정맥)에서 배아를 구분한다. </li><li> 주사 다음, 배아는 염료가 배아를 통해 순환되도록 2~3분에 대한 실온에서 PBS에 남아있을 수 있습니다. </li></ol> 2. 피부 vasculature 전체 마운트 분석 <ol><li> 염료는 배아에 걸쳐 순환 수 있도록 후, 다시, 사지의 지역에서 피부 샘플을 제거하고 복부 등 8,9. </li><li> 차가운 PBS의 피부 샘플을 씻고, 그리고 이시간 8,9에 대한 4% PFA / PBS에 수정. 십분 2mL 차가운 PBS로 4mL 명확 약병에 각각에 대해 3 번 씻으십시오. </li><li> 현미경에 놓습니다 스킨 샘플 100 각 슬라이드에 미디어를 장착의 μl와 커버 유리 슬라이드 및 추가합니다. </li><li> 슬라이드는 어두운 저장 영역에서 건조하도록 허용합니다. </li><li> 2 &#39;이미지 수집&#39;섹션 단계로 진행합니다. </li></ol> 3. thymus와 심장 vasculature 및 cryosections에 대한 perivascular 세포의 멀티 컬러 라벨은 (제 1, 7 단계에서 계속) <ol><li> 액체 질소에서 &#39;플래시 프리즈&#39;전체 배아. 공룡의 태아가되며 -80 ° C에서 분석까지 저장할 수 있습니다. </li><li> 또는, thymus을 해부하다 4 ° C PBS로 씻어, 2 시간 동안 2mL 4 % Paraformaldehyde (PFA) / PBS에 수정. -80에서 사용 ° C. 때까지 차가운 PBS, 10에서 개최 thymi하고, 동결 및 상점에서 10 분 동안 3 번 씻으십시오 </li><li> cryosectioning 들어, 섹션에서 &#39;블록&#39;에 OCT를 확산하고 sectioning에 대한 배아 또는 해부 기관 / 티슈를 탑재합니다. </li><li> 10 μm의 두께는 섹션으로 냉동 조직을 잘라 슬라이드에 수집합니다. </li><li> 50~10분위한 아세톤의 섹션을 수정. 추운 TBS에서 3 번 씻으십시오. </li><li> 실온에서 습도 챔버에서 10 % 당나귀 혈청 / TBS에 차단합니다. </li><li> 4 습도 챔버의 주요 항체 100 μl 1 1 시간 하루에 대한 섹션을 품어 ° C : 예제에서, 우리는 쥐의 반 마우스 CD31 (1:100) 라벨 내피하고, 염소 반 마우스 PDGFR - β를 사용하여 (1:100) perivascular 세포를 분류합니다. 이것은 항체가 균일 섹션에 걸쳐 확산되도록하기 위해 개별적으로 절단 Parafilm 스트립과 함께 슬라이드를 커버하는 데 유용합니다. </li><li> 주요 항체와 부화 다음, 차가운 TBS에서 섹션에게 3 회 씻는다. 최소 30 분 적절한 보조 항체의 100 μl로 품어. </li><li> 추운 TBS에서 3 번 씻으십시오. 100 각 슬라이드에 미디어를 장착의 μl와 덮개 유리를 추가합니다. </li><li> 슬라이드는 어두운 저장 영역에서 건조하도록 허용합니다. </li><li> &#39;이미지 수집&#39;섹션을 참조하십시오. </li></ol> 4. thymus vasculature 및 vibratome 섹션에 대한 perivascular 세포의 멀티 컬러 라벨은 (제 1, 7 단계에서 계속) <ol><li> 배아로부터 thymus 엽 (叶)을 해부하다 차가운 PBS로 린스. </li><li> 2 시간 동안 실온에서 4% PFA / PBS에 thymus을 수정. </li><li> 용융 낮은 4%의 작은 플라스틱 카트리지와 잠수함의 PBS - 트리톤 X (0.15 %) 3 시간, 10 분 장소 thymi에 씻으 아가로 오스 / PBS (~ 4 ° C). thymus은 카트리지의 하단에 접촉해야합니다. </li><li> 아가로 오스는 얼음 (3~5분)에 응고 수 있습니다. 초과 아가로 오스를 잘라하기 위해 면도날을 사용합니다. vibratome 블록에 접착제를 추가하고 차단하는 샘플을 준수합니다. </li><li> 샘플 및 블레이드가 포장되어 때까지 vibratome 물을 욕조에 차가운 PBS를 추가합니다. </li><li> 속도와 진폭을 (높은 진폭과 낮은 중간 속도 소프트 thymus 섹션에 이상적입니다) 설정합니다. 섹션 초과 교반으로 인해 헤어질 경우 진폭은 감소한다. </li><li> 50 음 섹션을 잘라 버릴거야. </li><li> 붓을를 사용 차가운 PBS에 24 잘 microplate에 섹션을 수집합니다. </li><li> 30 분 동안 PBS - 트리톤 X에서 10 % 당나귀 혈청 (0.15 %) 500 μl의 블록 섹션. </li><li> 같은 차 항체와 함께 하루를 8 시간에 대해 품어 섹션 안티 CD31 및 안티 - PDGFR - β, 대상 24 잘 microplate 인치 </li><li> 4 8 시간 총 ° C.여 PBS - 트리톤 X 3 회 (0.15 %)를 씻으십시오 </li><li> 30 분 동안 PBS - 트리톤 X에서 10 % 당나귀 혈청 (0.15 %)에서 블록 섹션. </li><li> 4 ° C와 함께 적절한 보조 항체에 하루에 8 시간 섹션을 품어. </li><li> 4 8 시간 총 ° C.여 PBS - 트리톤 X 3 회 (0.15 %)를 씻으십시오 </li><li> 다시 수정 샘플 4%에 PFA / 얼음에 30 분 PBS. </li><li> 얼음 30 분 이상의 PBS - 트리톤 X 3 회 (0.15 %)를 씻으십시오. </li><li> 25% MeOH, 50 % MeOH, 75 % MeOH, 각 단계를위한 10 분 100 % MeOH : 등급 MeOH / PBS - 트리톤 X 시리즈를 통해 샘플을 탈수. 십분 후에 신선한 MeOH로 100 % MeOH를 교체하고 실온에서 1 시간 품어. </li><li> 유리 용기에서 혼합 BABB (벤질 알코올 : 벤질 벤조 산) 1시 2분 비율 인치 50 % BABB와 50 % MeOH의 최종 농도에 대한 MeOH와 BABB을 결합합니다. 10-15분에 대한 MeOH : BABB에서 샘플을 품어. </li><li> 상온에서, 10-15 분 또는 정리 할때까지 백퍼센트 BABB과 부화로 유리 용기 샘플을 전송합니다. </li><li> 측면으로 신선한 100 % BABB 및 전송 샘플과 우울증 슬라이드 (0.7mm 깊이)를 입력합니다. 매니큐어로부터 2-3 코트와 유리 (제 1.5) 및 인감을 커버 추가합니다. 상온에서 어둠의 강화 매니큐어가 손톱 허용 후 4 샘플을 저장 ° C. 참고 : 슬라이드가 완전히 공촛점 이미지 수집하기 전에 봉인해야합니다. 형광 염료가 BABB에 페이드 수있는 이미지는 12~24시간 이내에 취득해야합니다. </li><li> &#39;이미지 수집&#39;섹션을 참조하십시오. </li></ol> 5. 이미지 수집 <ol><li> 이미지 10 488과 계획 - 고차 색지움 20X/0.8 목표를 (512 X 512 픽셀)를 사용 공촛점 현미경으로 μm의 냉동 섹션 - (FITC-dextran/GSL 1 - B 사 isolectin), 543 -, 그리고 633 - NM 레이저 라인. </li><li> 공촛점 취득 전체 마운트 피부 및 50 μm의 488과 계획 - 고차 색지움 10X/0.4 목표를 (512 X 512 픽셀)를 사용하여 아가로 오스 - 임베디드 섹션의 Z - 섹션 - (FITC-dextran/GSL 1 - B 사 isolectin), 543 - 와 633 - nm의 레이저 라인. 시리얼 Z - 부분은 각 각 채널에 대해 1 미크론에서 순차적으로 수령해야합니다. </li><li> 자이스 혈구 Axiovision 4.6이나 기타 이미지 분석 소프트웨어를 사용하여 Z - 섹션을 직렬 재구성. </li></ol> 6. 대표 결과 : 배아 vasculature의 효율적인 라벨링은 배아 생쥐에서 혈관 결함을 평가하기위한 중요합니다. 그림 1은 각각 좌우 심실의 얼룩 (1E - F) 이외에 E16.5 thymus 혈관 (1A - B)와 CD31 (1B)와 함께 공동 상표, 특정 라벨을 보여줍니다. GSL I - isolectin B와 같은 섹션 1, 3, 5에 설명된 cryosections 4 프로토콜은 이러한 실험에 사용되었다. 전체 마운트 E16.5 생쥐의 피부 혈관 네트워크의 레이블, 섹션 1, 2에서 설명된 프로토콜을 사용하여, 5은 그림 1C - D에 표시됩니다. <img alt="그림 1" src="/files/ftp_upload/3267/3267fig1.jpg" /> 한 전설을 그림. FITC GSL I - E16.5 마우스 배아에 isolectin B 네 얼굴 정맥 주사. 배아 피부 vasculature 다음 주입의 주입 다음 배아 thymus의 A.의 Cryosection. isolectin B 4 CD31 공동 상표의 B. 병합합니다. C.와 D. 전체 마운트. E. 및 배아 심장의 E. F.의 Cryosection (오른쪽 뇌실) F. (뇌실 왼쪽) 다음과 같은 분사.

<ol>
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관심 없음 충돌 선언하지 않습니다.

섹션에서 전체 마운트 및 PECAM - 1 (CD31) 얼룩은 배아 생쥐에서 vasculature을 라벨에 대한 종래의 방법입니다. 이 방법은 직접 및 / 또는 간접 immunofluorescence와 마우스 조직을 permeabilize하기 위해 세제의 사용을 필요로합니다. 이것은 다소 적시에 처리하는 증명한다. 여기, 우리는 FITC - dextran 또는 isolectin B 사 안면 정맥 주사 직접함으로써 항체 라벨 단계에 대한 요구를 제거, 배아 vasculature 레이?...

<table border="1"><tbody><tr><th> 시약의 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> FITC - dextran </td><td> 시그마 </td><td> FD150S - 1G </td></tr><tr><td> isolectin B 4 - 플루오레신는 GSL 1 분류 </td><td> 벡터 연구소 </td><td> FL - 1201 </td></tr><tr><td> 빠른 그린 </td><td> MP의 Biomedicals </td><td> 195,178 </td></tr><tr><td> PFA </td><td> Fluka </td><td> 76,240 </td></tr><tr><td> 태아 소 혈청 </td><td> 애틀랜타 체액 </td><td> S11550 </td></tr><tr><td> 최적의 절삭 온도 컴파 운드 (OCT </td><td> VWR </td><td> 25608-930 </td></tr><tr><td> 아세톤 </td><td> JT 베이커 </td><td> 9006-33 </td></tr><tr><td> 당나귀 세럼 </td><td> 잭슨 </td><td> 017-000-121 </td></tr><tr><td> 쥐 방지 마우스 CD31, </td><td> BD Pharmingen </td><td> 558,736 </td></tr><tr><td> 염소 안티 마우스 PDGFR - β </td><td> R &amp; D 시스템 </td><td> AF1042 </td></tr><tr><td> 당나귀 안티 쥐 CD31 알렉사 647 (Invitrogen) </td><td> Biolegend </td><td> 102,516 </td></tr><tr><td> 당나귀 안티 염소 알렉사 594 (Invitrogen) </td><td> Invitrogen </td><td> A11058 </td></tr><tr><td> 트리톤 X -100 </td><td> 시그마 - 올드 리치 </td><td> X - 100 </td></tr><tr><td> 낮은 용융 아가로 오스 / PBS </td><td> 시그마 - 올드 리치 </td><td> A9414 - 25G </td></tr><tr><td> 메탄올 </td><td> 피셔 과학 </td><td> A413 - 4 </td></tr><tr><td> 벤질 알코올 </td><td> Acros 과학 </td><td> 148,390,010 </td></tr><tr><td> 벤질 벤조 산 </td><td> Acros 과학 </td><td> 105,860,010 </td></tr><tr><td> 우울증 슬라이드 </td><td> 피셔 과학 </td><td> S175201 </td></tr><tr><td> Fluorogel </td><td> 전자 현미경 과학 </td><td> 17985-10 </td></tr><tr><td> 커버 유리 (22X22) -1.5 </td><td> 써모 과학 </td><td> 152,222 </td></tr><tr><td> 자이스 혈구 LSM 510 메타 공촛점 현미경 </td><td> 자이스 혈구 </td><td></td></tr><tr><td> 마이크로 해부 포셉 </td><td> Roboz </td><td> RS - 5135 </td></tr><tr><td> Parafilm 번호 OM992 </td><td> 피셔 과학 </td><td> 13-374-16 </td></tr><tr><td> 12 24 잘 microplates </td><td> 에버그린 과학 </td><td> 222-8044 - 01F </td></tr><tr><td> Superfrost / 플러스 현미경 슬라이드 </td><td> 피셔 과학 </td><td> 12-550-15 </td></tr><tr><td> 4mL 맑은 튜브 </td><td> 국립 과학 </td><td> B7800 - 2 </td></tr></tbody></table>

a method for labeling vasculature in embryonic mice

기능성 혈관 네트워크의 설립 organogenesis의 필수적인 부분이며, 최적의 장기의 기능이 필요합니다. 예를 들어, thymus 적절한 vasculature 형성과 patterning에있는 기관 및 주변에 성숙한 T 세포 출구로 thymocyte 입학 필수적입니다. thymus에서 혈관의 공간적 배열은 로컬 microenvironment의 신호, 즉 상피 세포 thymic (TEC)에 의존적일 수 밖에 없습니다. 몇 가지 최근의 보고서는 thymus 혈관 결함 1,2에서 이러한 신호 결과의 중단을 제안한다. 이전 연구는 신생아와 성인의 thymus vasculature 1,2 레이블을하는 데 사용되는 기술을 설명합니다. 우리는 여기서 배아 thymus에서 혈관을 라벨에 대한 기술을 보여줍니다. 이 방법은 FITC - dextran 또는 Griffonia (Bandeiraea) Simplicifolia 렉틴 I (GSL 1 - isolectin B 4)의 사용을 결합한 얼굴 정맥 주사 및 CD31 항체 얼룩 thymus 혈관 구조와 thymic perivascular mesenchyme 3-5 라벨 PDGFR - β를 식별할 수 있습니다. cryosections 또는 vibratome 섹션을 사용할 수있는 옵션도 제공됩니다. 이 프로토콜은 thymus 혈관 형성에 TEC - 파생 분자의 역할을 정의하는 것이 중요합니다 thymus 혈관 결함을 식별하는 데 사용할 수 있습니다. 방법은 전체 vasculature를 레이블로, 그것은 또한 피부와 마음 60-10 포함한 배아에 걸쳐 여러 기관과 조직에 혈관 네트워크를 분석하는 데 사용할 수 있습니다.

Watch this Scientific Journal Video about A Method for Labeling Vasculature in Embryonic Mice at JoVE.com

A Method for Labeling Vasculature in Embryonic Mice

이 문서는 배아 피부와 thymus 혈관을 라벨에 대한 방법을 설명합니다.

배아 마우스에 Vasculature을 라벨링하는 방법

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

a-method-for-labeling-vasculature-in-embryonic-mice

Research

JoVE Journal

Immunology and Infection

15.5K 조회수.  University of Georgia. 이 문서는 배아 피부와 thymus 혈관을 라벨에 대한 방법을 설명합니다.

비디오: A Method for Labeling Vasculature in Embryonic Mice

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

사이 Synergism을 맡은 이후로 발광 생물 이미징을 사용하여 연쇄상 구균 pneumoniae</em유아 마우스에서>와 인플루엔자 바이러스

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

연구

면역학 및 감염병학

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3의 HIV - 1 Tropism의 Genotypic 추론 사용 인구를 기반 시퀀싱

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

알렉사 플루어 라벨링을 통해 뎅그열 바이러스를 떠올리

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Non-invasive Optical Imaging of the Lymphatic Vasculature of a Mouse

The lymphatic vascular system is an important component of the circulatory system that maintains fluid homeostasis, provides immune surveillance, and mediates fat absorption in the gut. Yet despite its critical function, there is comparatively little understanding of how the lymphatic system adapts to serve these functions in health and disease1. Recently, we have demonstrated the ability to dynamically image lymphatic architecture and lymph "pumping" action in normal human subjects as well as in persons suffering lymphatic dysfunction using trace administration of a near-infrared fluorescent (NIRF) dye and a custom, Gen III-intensified imaging system2-4. NIRF imaging showed dramatic changes in lymphatic architecture and function with human disease. It remains unclear how these changes occur and new animal models are being developed to elucidate their genetic and molecular basis. In this protocol, we present NIRF lymphatic, small animal imaging5,6 using indocyanine green (ICG), a dye that has been used for 50 years in humans7, and a NIRF dye-labeled cyclic albumin binding domain (cABD-IRDye800) peptide that preferentially binds mouse and human albumin8. Approximately 5.5 times brighter than ICG, cABD-IRDye800 has a similar lymphatic clearance profile and can be injected in smaller doses than ICG to achieve sufficient NIRF signals for imaging8. Because both cABD-IRDye800 and ICG bind to albumin in the interstitial space8, they both may depict active protein transport into and within the lymphatics. Intradermal (ID) injections (5-50 &mu;l) of ICG (645 &mu;M) or cABD-IRDye800 (200 &mu;M) in saline are administered to the dorsal aspect of each hind paw and/or the left and right side of the base of the tail of an isoflurane-anesthetized mouse. The resulting dye concentration in the animal is 83-1,250 &mu;g/kg for ICG or 113-1,700 &mu;g/kg for cABD-IRDye800. Immediately following injections, functional lymphatic imaging is conducted for up to 1 hr using a customized, small animal NIRF imaging system. Whole animal spatial resolution can depict fluorescent lymphatic vessels of 100 microns or less, and images of structures up to 3 cm in depth can be acquired9. Images are acquired using V++ software and analyzed using ImageJ or MATLAB software. During analysis, consecutive regions of interest (ROIs) encompassing the entire vessel diameter are drawn along a given lymph vessel. The dimensions for each ROI are kept constant for a given vessel and NIRF intensity is measured for each ROI to quantitatively assess "packets" of lymph moving through vessels.

Recently developed imaging techniques using near-infrared fluorescence (NIRF) may help elucidate the role the lymphatic system plays in cancer metastasis, immune response, wound repair, and other lymphatic-associated diseases.

마우스의 림프 Vasculature의 비 침습적 광학 이미징

The lymphatic  vascular system is an important component of the circulatory system that  maintains fluid homeostasis, provides immune surveillance, and ...

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

This protocol outlines the steps required to longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging tomography with integrated &mu;CT (DLIT-&mu;CT) and the subsequent use of this data to generate a four dimensional (4D) movie of the infection cycle. To develop the 4D infection movies and to validate the DLIT-&mu;CT imaging for bacterial infection studies using an IVIS Spectrum CT, we used infection with bioluminescent C. rodentium, which causes self-limiting colitis in mice. In this protocol, we outline the infection of mice with bioluminescent C. rodentium and non-invasive monitoring of colonization by daily DLIT-&mu;CT imaging and bacterial enumeration from feces for 8 days.
The use of the IVIS Spectrum CT facilitates seamless co-registration of optical and &mu;CT scans using a single imaging platform. The low dose &mu;CT modality enables the imaging of mice at multiple time points during infection, providing detailed anatomical localization of bioluminescent bacterial foci in 3D without causing artifacts from the cumulative radiation. Importantly, the 4D movies of infected mice provide a powerful analytical tool to monitor bacterial colonization dynamics in vivo.

4D Multimodality Imaging of Citrobacter rodentium Infections in Mice

Multi-modality imaging is a valuable approach for studying bacterial colonization in small animal models. This protocol outlines infection of mice with bioluminescent Citrobacter rodentium and the longitudinal monitoring of bacterial colonization using composite 3D diffuse light imaging tomography with &mu;CT imaging to create a 4D movie of C. rodentium infection.

의 4D 좌석 Multimodality 이미징<em&gt; Citrobacter rodentium</em마우스에서&gt; 감염

This protocol outlines the steps required to  longitudinally monitor a bioluminescent bacterial infection using composite 3D diffuse light imaging ...

A Non-invasive and Technically Non-intensive Method for Induction and Phenotyping of Experimental Bacterial Pneumonia in Mice

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant preclinical advances in our understanding of the underlying cellular and molecular pathogenesis. In vivo mouse models capture the integrated physiology and resilience of the host defense response in a manner not revealed by alternative, simplified ex vivo approaches. Several methods have been described in the literature for intrapulmonary inoculation of bacteria in mice, including aerosolization, intranasal delivery, peroral endotracheal cannulation under &#39;blind&#39; and visualized conditions, and transcutaneous endotracheal cannulation. All methods have relative merits and limitations. Herein, we describe in detail a non-invasive, technically non-intensive, inexpensive, and rapid method for intratracheal delivery of bacteria that involves aspiration (i.e., inhalation) by the mouse of an infectious inoculum pipetted into the oropharynx while under general anesthesia. This method can be used for pulmonary delivery of a wide variety of non-caustic biological and chemical agents, and is relatively easy to learn, even for laboratories with minimal prior experience with pulmonary procedures. In addition to describing the aspiration pneumonia method, we also provide step-by-step procedures for assaying the subsequent in vivo pulmonary innate immune response of the mouse, in particular, methods for quantifying bacterial clearance and the cellular immune response of the infected airway. This integrated and simple approach to pneumonia assessment allows for rapid and robust evaluation of the effect of genetic and environmental manipulations upon pulmonary innate immunity.

Several methods have been described in the literature for modeling bacterial pneumonia in mice. Herein, we describe a non-invasive, inexpensive, rapid method for inducing pneumonia via aspiration (i.e., inhalation) of a bacterial inoculum pipetted into the oropharynx. Downstream methods for assessment of the pulmonary innate immune response are also detailed.

마우스의 실험 세균성 폐렴의 유도 및 표현형에 대한 비 침습적 및 기술적 비 집약적 인 방법

Although community-acquired pneumonia remains a major public health problem, murine models of bacterial pneumonia have recently facilitated significant ...

Induction of Paralysis and Visual System Injury in Mice by T Cells Specific for Neuromyelitis Optica Autoantigen Aquaporin-4

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human central nervous system (CNS) autoimmune demyelinating disease, creation of an AQP4-targeted model with both clinical and histologic manifestations of CNS autoimmunity has proven challenging. Immunization of wild-type (WT) mice with AQP4 peptides elicited T cell proliferation, although those T cells could not transfer disease to na&#239;ve recipient mice. Recently, two novel AQP4 T cell epitopes, peptide (p) 135-153 and p201-220, were identified when studying immune responses to AQP4 in AQP4-deficient (AQP4-/-) mice, suggesting T cell reactivity to these epitopes is normally controlled by thymic negative selection. AQP4-/- Th17 polarized T cells primed to either p135-153 or p201-220 induced paralysis in recipient WT mice, that was associated with predominantly leptomeningeal inflammation of the spinal cord and optic nerves. Inflammation surrounding optic nerves and involvement of the inner retinal layers (IRL) were manifested by changes in serial optical coherence tomography (OCT). Here, we illustrate the approaches used to create this new in vivo model of AQP4-targeted CNS autoimmunity (ATCA), which can now be employed to study mechanisms that permit development of pathogenic AQP4-specific T cells and how they may cooperate with B cells in NMO pathogenesis.

Here, we present a protocol to induce paralysis and opticospinal inflammation by transfer of aquaporin-4 (AQP4)-specific T cells from AQP4-/- mice into WT mice. In addition, we demonstrate how to use serial optical coherence tomography to monitor visual system dysfunction.

마비 및 비주얼 시스템 부상 Neuromyelitis Optica Autoantigen Aquaporin-4에 대 한 T 세포 관련 하 여 쥐에서의 유도

While it is recognized that aquaporin-4 (AQP4)-specific T cells and antibodies participate in the pathogenesis of neuromyelitis optica (NMO), a human ...

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 ...

Deep Vein Thrombosis Induced by Stasis in Mice Monitored by High Frequency Ultrasonography

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death through pulmonary embolism or results in the post-thrombotic syndrome, characterized by chronic leg pain, swelling, and ulceration, or in chronic pulmonary hypertension resulting in significant chronic respiratory compromise. This is the most common cardiovascular disease after myocardial infarction and ischemic stroke and is a clinical challenge for all medical disciplines, as it can complicate the course of other disorders such as cancer, systemic disease, surgery, and major trauma.
Experimental models are necessary to study these mechanisms. The stasis model induces consistent thrombus size and a quantifiable amount of thrombus. However, it is necessary to systematically ligate side branches of the inferior vena cava to avoid variability in thrombus sizes and any erroneous data interpretation. We have developed a non-invasive technique to measure thrombus size using ultrasonography. Using this technique, we can assess thrombus development and resolution over time in the same animal. This approach limits the number of mice required for quantification of venous thrombosis consistent with the principle of replacement, reduction, and refinement of animals in research. We have demonstrated that thrombus weight and histological analysis of thrombus size correlate with measurement obtained with ultrasonography. Therefore, the current study describes how to induce deep vein thrombosis in mice using the inferior vena cava stasis model and how to monitor it using high frequency ultrasound.

The present protocol describes the steps to obtain venous thrombosis using a stasis model. In addition, we are using a non-invasive method to measure thrombus formation and resolution over time.

스테이 시스 고주파 초음파에 의해 모니터링 하는 쥐에 의해 유도 된 깊은 정 맥 혈전 증

Venous thrombosis is a common condition affecting 1 - 2% of the population, with an annual incidence of 1 in 500. Venous thrombosis can lead to death ...

A Silver Nanoparticle Method for Ameliorating Biliary Atresia Syndrome in Mice

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections may be one possible cause. The typical animal model used for studying BA is established by inoculating a neonatal mouse with a rhesus rotavirus. Silver nanoparticles have been shown to exert antibacterial and antiviral effects; their function in the BA mouse model is evaluated in this study. Currently, in BA animal experiments, the methods used to improve the symptoms of BA mice are generally symptomatic treatments given via food or other drugs. The aim of this study is to demonstrate a new method for ameliorating BA syndrome in mice by the intraperitoneal injection of silver nanoparticles and to provide detailed methods for preparing the silver nanoparticle gel formulation. This method is simple and widely applicable and can be used to research the mechanism of BA, as well as in clinical treatments. Based on the BA mouse model, when the mice exhibit jaundice, the prepared silver nanoparticle gel is injected intraperitoneally to the surface of the lower liver. The survival status is observed, and biochemical indicators and liver histopathology are examined. This method allows a more intuitive understanding of both the establishment of the BA model and novel BA treatments.

This article describes in detail a method based on silver nanoparticles for ameliorating biliary atresia syndrome in an experimental biliary atresia mouse model. A solid understanding of the reagent preparation process and the neonatal mouse injection technique will help familiarize researchers with the method used in neonatal mouse model studies.

쥐에서 담 즙이 폐쇄 증 증후군 장황 하는 실버 나노 방법

Biliary atresia (BA) is a severe type of cholangitis with high mortality in children of which the etiology is still not fully understood. Viral infections ...

Methods Article

배아 마우스에 Vasculature을 라벨링하는 방법