Os autores agradecem ao Dr. Le Trinh e Dr. Tobin para generosamente fornecer a α-catenina: linha citrino peixes e Bill Jackman por nos permitir fazer a filmagem em seu laboratório. Os autores reconhecem as fontes de financiamento Institutos Nacionais de Saúde (Grants 5P20RR016463, 8P20GM103423 e R15AI094406) e USDA (Projeto nº ME0-H-1-00517-13). Este manuscrito é publicado como principal Agricultura e Florestas Experiment Station número de publicação 3371.

NOTA: Todos os protocolos de cuidados de peixe-zebra e experimentos foram realizados de acordo com as diretrizes do NIH sob Animal Care Institucional e Comitê de Uso (IACUC) A2012-11-03 protocolo. 1. Zebrafish Elevando a 4 dias após a fertilização <ol><li> Recolhe AB peixe-zebra, ou quaisquer outras linhas transgénicas, dentro da primeira hora após a fertilização 3, como mostrado em outro vídeo 34. </li><li> Incubar 120 ovos em um 15 centímetros placas de Petri contendo 150 ml de mídia E3 (mM NaCl 5; KCl 0,17; 0,33 mM CaCl 2; 0,33 mM MgCl2; HEPES 2 mM; pH 6,8) com azul de metileno (0,3 ug / ml definitiva concentração) em um 33 ° C incubadora com 12/12 luz / fotoperíodo escuro. </li><li> Substituir o material após 6 h com E3 + PTU (1-fenil-2-tioureia, 10 ug / ml de concentração final) e remover os ovos mortos. </li><li> Incubar durante 4 dias a 33 ° C, trocando a mídia com nova E3 + PTU após 2 dias. </li></ol> 2. Eminjeção Micro-agulha Preparação <ol><li> Puxe capilar borosilicato (OD 1,2 milímetros; ID 0,69 milímetros) em um micro-agulha usando um extrator agulha com as seguintes configurações (550 Calor; 0 Pull; 130 Velocity; 110 Time). NOTA: As configurações irão variar de filamento para filamento para se obter micro-agulhas em forma semelhante (ver Figura 1) e necessitam de ser reajustados sempre que um novo filamento está instalado. </li><li> Encha uma micro-agulha com 3 ul de 0,4 um filtrou-tampão salino de fosfato (PBS; NaCl 137 mM; KCl 2,7 mM; 10 mM de Na 2 HPO 4; 1,8 mM de KH 2 PO 4, pH 7,4) utilizando uma pipeta de 20 uL microloader ponta. </li><li> Defina-se a micro-agulha num suporte de micropipeta em um micromanipulador ligado a um sistema de injecção de pressão. Ajuste a pressão de injeção para 30 PSI e a duração do pulso de 30 ms. </li><li> Usando uma placa de Petri com água destilada, inferior a micro-agulha até 1/5 da agulha está na água. Clipe da wi micro-agulhath uma pinça fina logo abaixo do nível da água. Traga a micro-agulha para fora da água e empurrar o pedal várias vezes até um bolus aparece. </li><li> Medir o diâmetro do bolo baixando a micro-agulha em um hemocitómetro em camadas com uma gota de óleo de tipo A imersão. Ajustar a duração do impulso para obter o volume desejado (ver Tabela 1). </li><li> Ajuste da contrapressão para o bólus se mantenha estável em óleo (diminuir a pressão, se aumenta o volume do bolo e aumentar a pressão de retorno se o volume de bolus diminui). Grave a duração do pulso para cada micro-agulha. </li><li> Retire a PBS da micro-agulha, aspirando-lo usando uma ponteira microloader e expulsar a esquerda-over PBS, colocando-o de volta no injector e apertar o interruptor de pulso contínua. Reserve cada micro-agulha. </li></ol> 3. Preparação Candida <ol><li> Streak Candida albicans transformada com otimizado-códon dTomato, GFP, BFP, EOS ou FarVermelho de um lote congelado (-80 ° C estoque em glicerol a 25%) usando um passador de madeira estéril para uma levedura dextrose peptine (YPD) placa de agar (10 g de extracto / L de levedura, 20 g / L de peptona, 20 g / L de dextrose , 20 g / L de agar; autoclavado e verteu-se 25 ml em placas de Petri) e incubar durante a noite a 30 ° C. </li><li> Escolha uma colónia utilizando um pino de madeira e estéril para inocular 5 ml de meio YPD líquido (10 g de extracto / L de levedura, 20 g / L de peptona, 20 g / L de dextrose, autoclavado e assepticamente em alíquotas de 5 ml em 16 x 150 milímetros de cultura de vidro tubos). </li><li> Incubar durante a noite a 30 ° C num tambor de rolos a 60 rpm. </li><li> Colete 1 ml de C. albicans cultura e transferência para um tubo de centrífuga de 1,7 ml estéril. </li><li> Centrifugar a 5000 xg por um par de segundos. Esvazie o sobrenadante e adicionar 1 ml de PBS estéril, vortex completamente. Repita isso duas vezes. </li><li> Contar as colónias utilizando um hemocitómetro por diluição do stock 1 mL de 1: 1000 em PBS estéril. </li></ol> 4. Injetar Zebrafish ema bexiga natatória <ol><li> Aquecer um prato de injecção (2% de agarose) durante 30 min em uma incubadora de 33 ° C. </li><li> No momento desejado, remover 100 dos 150 ml de E3 + UTP a partir do prato de 15 centímetros de Petri contendo o peixe para injectar, utilizando uma pipeta de 25 ml sem aspirar qualquer peixe-zebra. </li><li> Anestesiar 4 dpf peixe-zebra por adição de 2 ml de uma mistura 4 mg / ml tamponado tricaina metano sulfonato estoque (TR) para os restantes 50 ml de meio (concentração final de 200 ug / ml) e aguardar durante 15 minutos. </li><li> Sob um microscópio de dissecação, selecione peixe com uma bexiga natatória inflado. Os peixes também podem ser rastreados na época para fenótipo mais homogêneo, como a distribuição de neutrófilos em mpo: linha GFP utilizando um microscópio de epifluorescência dissecando. </li><li> Dilui-se a C. albicans de estoque a sua concentração original (passo 3.8) e 1,5 x 10 7 unidades formadoras de colónias por ml (cfu / ml) em PBS. </li><li> Transferir 30 peixes-zebra para o prato de injecção usando um tubo de transferência de plásticotte e remover o excesso de mídia. </li><li> Usando uma ferramenta de fio de pesca (0,012 polegadas de diâmetro linha de pesca super-colado em um capilar de borosilicato), fazer 3 linhas de 10 peixes, segurando o prato injeção vertical e orientar o peixe verticalmente. </li><li> Retire o excesso de mídia. </li><li> Absolutamente vortex do tubo com 1,5 x 10 7 ufc / mL C. albicans. </li><li> Encha um micro-agulha com 3 l de C. albicans utilizando uma nova ponta microloader pipeta e definir a duração do pulso para a definição apropriada (veja o passo 2.9). </li><li> Rode o prato de injeção para fazer um ângulo de 20 ° entre o micro-agulha ea cabeça do peixe, que aponta para a parte de trás da bexiga natatória (Figura 3A). </li><li> Empurrar o micro-agulha para dentro da bexiga natatória, certificando-se o furo da agulha está no lúmen, e uma vez que pressiona o pedal. </li><li> Repita o procedimento para cada peixe e transfira para um prato de recuperação (25 ml E3 + PTU) inundando o prato com um E3d esvaziando-a no prato de recuperação. </li><li> Transferir mais 30 peixes para o prato de injecção. </li><li> Repita injeção com um novo micro-agulha cheia com cuidadosamente agitadas C. albicans. </li><li> Concluir através da injecção do controlo de PBS, se necessário, utilizando um prato de injecção diferente, para evitar a contaminação e transferir para uma cápsula de recuperação separada (passo 4.10). </li></ol> 5. Triagem do Peixe para o inóculo desejado <ol><li> Preparar 40 ml de 0,4% de baixo ponto de fusão solução de agarose (LMA) através da adição de 160 mg de agarose de baixo ponto de fusão a 40 ml de E3, fervendo por microondas até à dissolução, e de se arrefecer para 37 ° C. Adicionar 400 ul de TR (concentração final de 200 ug / ml) uma vez arrefecida. </li><li> Depois de 30 minutos de recuperação, anestesiar os peixes como descrito (passo 4.3 utilizando apenas 1 ml de TR em 25 mL E3 + PTU, concentração final de 200 ug / ml). </li><li> 30 Transferência de peixe (com um mínimo de meios quanto possível) a 2 ml de LMA em peso de um barco. </li><li> Aspirar peixes individuais com uma pipeta de transferência e a placa de peixe com um LMA em gotas de uma placa de imagiologia de 96 poços, utilizando-se apenas os 10 x 6 poços centrais. </li><li> Repita até que todos os peixes para a tela são carregados para a placa de imagem. </li><li> Deixar os meios solidificar à temperatura ambiente durante 5 min. </li><li> Coloque o peixe do seu lado, certificando-se que eles estão em contato com o fundo de vidro. </li><li> Usando o objetivo 20X em um epifluorescência / microscópio confocal invertido e o filtro apropriado, registrar o número de C. células de levedura albicans na bexiga natatória de cada peixe. </li><li> Selecione o peixe com 15-25 células de levedura na bexiga natatória (ou inóculo desejado), eutanásia peixe não selecionados (800 ug / ml de TR na E3) e retornar peixe selecionado para um prato fundo Petri com 50 ml de E3 + PTU por adicionar 3 gotas de E3 para a chapa de imagem poços selecionados e aspirar o peixe com uma pipeta de plástico. </li><li> Incubar a 33 ° C durante o tempo desejado. </li></ol> 6. Imaging o Peixe Pós-screening <ol><li> No momento desejado, remover 25 dos 50 ml de meio E3 + PTU do prato fundo Petri contendo o peixe para a imagem. </li><li> Anestesiar os peixes como descrito (passo 4.3 utilizando apenas 1 ml de TR em 25 ml E3 + PTU remanescente, concentração final de 200 ug / ml). </li><li> Placa o peixe para uma placa de imagiologia de 96 poços e posição apropriadamente (passos 5,3-5,7). </li><li> Imagem por microscopia confocal utilizando os lasers adequados e filtros de emissão. Primeiro foco nos peixes usando o objetivo 4X sob contraste de interferência diferencial (DIC) e adquirir imagens da região de interesse, utilizando o objetivo 20X com os lasers confocal em modo de digitalização com 2 velocidade ms / pixel e 1024 x 600 pixel proporção. Adquirir Z-stacks com 1 mm tamanho do passo, aplicando-se o modo de filtro de Kalman de 3 frames. </li><li> Retornar para um prato fundo de Petri com 50 ml E3 + PTU ou em individual (poços de 24 poços do prato de cultura, 3 ml E3 + PTU) e incubar a 33 ° C. </li></ol> 7. Dissecando a bexiga natatória <ol><li> Encher uma seringa de 10 ml com graxa alto vácuo. </li><li> Prepara-se uma LMA 2% (passo 5.3 com 160 mg de agarose a baixo ponto de fusão em 8 ml de E3). </li><li> No momento desejado, anestesiar o peixe (passo 5.2). </li><li> Transferir 10 de peixe para um tubo de centrífuga de 1,7 mL com 1 ml de E3 e eutanásia por adição de 200 ul de 4 mg / ml tricaina (concentração final de 800 ug / ml) para o tubo de centrifugação e incubação durante 15 min à temperatura ambiente. </li><li> Prepare uma lâmina de vidro de imagem, colocando 4 gotas de graxa de vácuo em uma praça, com 1 cm de diferença em um microlâmina (75 x 25 mm, Figura 2). </li><li> Coloque 2 gotas de 2% LMA no meio da praça. </li><li> Coloque um peixe em uma lâmina de vidro separada sob um microscópio de dissecação e verificar que o coração parou de bater, retire o excesso de água. </li><li&gt; Usando uma pinça fina dissecar a bexiga natatória, mantendo a cabeça em uma posição fixa com a pinça esquerda e puxando para baixo o intestino anterior com a pinça direita (Figura 4A). </li><li> Pegue o swimbladder pelo duto pneumático com a pinça esquerda, separando-o do aparelho digestivo, e colocá-lo no centro da LMA 2% antes de ela se solidifica. </li><li> Colocar uma tampa de deslizamento (18 x 18 mm) na parte superior da lâmina de vidro de imagem e empurrá-lo até que ela toque a graxa vácuo, bem como o LMA (Figura 2). </li><li> Imagem dentro de 10 min de esvaziamento, tal como descrito na etapa 6.4. Repita o passo 7,7-7,11 com os outros peixes. </li></ol>

The authors have nothing to disclose.

Os avanços e as limitações do modelo de doença microinjeção swimbladder O modelo apresentado aqui é uma extensão do modelo de imersão mucosa candidíase descrito no Gratacap et al (2013).; acrescenta as vantagens de um tempo de infecção controlada, uma dose de infecção altamente reprodutível, e, por conseguinte, uma maior eficiência. Nós demonstramos aqui novos métodos que permitem a documentação temporais não-invasivo de dinâmica de infecção em grande detalhe, b...

Mucosal infections can lead to life threatening bloodstream infections due to the damage of the epithelial barrier, which allows pathogens access to the systemic environment1,2. In addition, mucosal infections can also cause significant immunopathology even when contained externally3-5. The commensal unicellular fungus Candida albicans is present in the majority of the population in the oral cavity and other mucosal sites6-9. Although normally contained by innate and adaptive immune responses, innate immune defects and medical interventions can lead to severe mucosal candidiasis. The assault on the epithelial barrier results in an increased risk of life threatening disseminated disease as well as immunopathology, as in the case of vulvo-vaginal candidiasis, additionally C. albicans colonization has been linked with lung immune homeostasis10,11. Disseminated candidiasis is now the fourth most common bloodstream infection in intensive care units12 and mortality as high as 40% makes it a major concern. Due to the increase in immunomodulatory treatments for patients with autoimmune diseases, cancer or organ transplants, it is imperative to understand the interaction between this pathogen and the mucosal immune compartment.
The majority of cell biological advances regarding C. albicans-cell interactions at the mucosal level come from in vitro13-15 and murine models16-18. Both these approaches have distinct advantages, but the ability to image live cells at high resolution in an intact host has limited the temporal and spatial characterization of the infection. For these studies, there is the need for an in vivo model where the interaction of pathogen, innate immune and epithelial cells can be visualized in an intact vertebrate host.
The zebrafish has emerged as an invaluable tool for the understanding of human disease, mainly due to its transparency and amenability to genetic manipulation. Cell and organ development have been imaged in exquisite detail, which has led to the description of novel immune cell behaviors, such as T cell behavior in the developing thymus19 or the battle between intracellular mycobacteria and phagocytes20-22. Recent work has described intestinal microbe-host interactions in zebrafish and shown that microbial colonization of the intestinal tract affects host intestinal physiology and resistance to other infections23,24. Furthermore, infection through the gut epithelium has been described for several pathogens.
In contrast to the intestinal tract, the swimbladder represents a more isolated and complementary mucosal model. This organ is an extension of the developing gut tube and forms anteriorly to the liver and pancreas25,26. It produces surfactant, mucus and antimicrobial peptides27,28 and anatomically, as well as ontogenetically, this organ is considered a homologue of the mammalian lung29,30. Since the pneumatic duct remains connected to the gut in the zebrafish, this allows for immersion infection to occur naturally. Remarkably, the only known naturally occurring infections of fish with Candida species are C. albicans infections in the swimbladder31. We recently described an experimental immersion infection model where C. albicans infects the swimbladder, and found that this infection recapitulates some of the hallmarks of C. albicans-epithelial interaction in vitro32,33. 
In the method presented here, the original immersion infection model is improved by directly injecting C. albicans into the swimbladder of 4 days post fertilization (dpf) zebrafish. This allows for precise temporal control of infection as well as a highly reproducible inoculum. It permits detailed intravital imaging, coupled with the versatility of the zebrafish model. As an example of what can be done with this method, we present the spatio-temporal dynamics of C. albicans growth along with neutrophil recruitment to the site of infection. Because zebrafish swimbladder tissue is challenging to image intravitally, we also present a rapid swimbladder dissection technique that improves fluorescence signal and microscopic resolution. These methods expand the toolbox for fungal, immunological, and aquaculture research as well as describing a novel infection route that may be translated to model other fungal, bacterial or viral infections of mucosal surfaces.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:261px;">Name</td>
			<td style="width:259px;">Company</td>
			<td style="width:176px;">Catalog Number</td>
			<td style="width:88px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.7 mL tubes</td>
			<td>Axygen</td>
			<td>MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Deep Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>89107-632</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-7M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>VWR Scientific</td>
			<td>90000-726</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone</td>
			<td>VWR Scientific</td>
			<td>90000-264</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dextrose</td>
			<td>Fisher Scientific</td>
			<td>D16-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar</td>
			<td>VWR Scientific</td>
			<td>90000-760</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine tweezers (Dumont Dumoxel #5)</td>
			<td>Fine Science Tools</td>
			<td>11251-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wooden Dowels</td>
			<td>VWR Scientific</td>
			<td>10805-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Low Melt Agarose</td>
			<td>VWR Scientific</td>
			<td>12001-722</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flaming Brown Micropipette Puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate capillary</td>
			<td>Sutter Instruments</td>
			<td>BF120-69-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MPPI-3 Injection system</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPPI-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Back Pressure Unit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>BPU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette Holder kit</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MPIP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Foot Switch</td>
			<td>Applied Scientific Instrumentation</td>
			<td>FSW</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Applied Scientific Instrumentation</td>
			<td>MM33</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnetic Base</td>
			<td>Applied Scientific Instrumentation</td>
			<td>Magnetic Base</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tricaine methane sulfonate</td>
			<td>Western Chemical Inc.</td>
			<td>MS-222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dissecting Scope</td>
			<td>Olympus</td>
			<td colspan="2">SZ61 top SZX-ILLB2-100 base</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal Microscope</td>
			<td>Olympus</td>
			<td colspan="2">IX-81 with FV-1000 laser scanning confocal system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20x microscope objective</td>
			<td>Olympus</td>
			<td>UPlanSApo 20x/0.75</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Roller drum</td>
			<td>New Brunswick Scientific</td>
			<td>TC-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microloader pipette tips</td>
			<td>Eppendorf</td>
			<td>930001007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass culture tubes (16 x 150 mm)</td>
			<td>VWR Scientific</td>
			<td>60825-435</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>VWR Scientific</td>
			<td>BDH4534-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KCl</td>
			<td>VWR Scientific</td>
			<td>BDH4532-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgSO4</td>
			<td>VWR Scientific</td>
			<td>BDH0246-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES (Corning)</td>
			<td>VWR Scientific</td>
			<td>BDH4520-500GP</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Children clay (Play-Doh)</td>
			<td>Hasbro</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CaCl2</td>
			<td>Fisher Scientific</td>
			<td>C69-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methylene Blue</td>
			<td>VWR Scientific</td>
			<td>VW6276-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PTU</td>
			<td>Sigma</td>
			<td>P7629-10G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer (Hausser scientific)</td>
			<td>VWR Scientific</td>
			<td>15170-172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Type A immersion oil</td>
			<td>Blue Marble Products</td>
			<td>51935</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex Genie</td>
			<td>VWR Scientific</td>
			<td>14216-184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose (Lonza)</td>
			<td>VWR Scientific</td>
			<td>12001-870</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Na2HPO4</td>
			<td>Fisher Scientific</td>
			<td>S374-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">KH2PO4</td>
			<td>Fisher Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fishing wire</td>
			<td>Stren</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">96 well imaging plate (Sensoplate)</td>
			<td>Greiner Bio-One</td>
			<td>655892</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High vacuum grease (Dow Corning)</td>
			<td>VWR Scientific</td>
			<td>59344-055</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microslide (25 x 75 mm)</td>
			<td>VWR Scientific</td>
			<td>48300-025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover slips (18 x 18 mm), No 1.5</td>
			<td>VWR Scientific</td>
			<td>48366-045</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 cm Petri dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>32-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol (EMD chemicals)</td>
			<td>VWR Scientific</td>
			<td>EMGX0185-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well culture dish (Olympus plastics)</td>
			<td>Genesee Scientific</td>
			<td>25-107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Weight boats (8.9 cm)</td>
			<td>VWR Scientific</td>
			<td>89106-766</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling mucosal candidiasis in larval zebrafish by swimbladder injection

Defesa precoce contra patógenos da mucosa consiste de uma barreira epitelial e células imunitárias inatas. A imunocompetência de ambos, e sua intercomunicação, são de extrema importância para a proteção contra infecções. As interações de células imunitárias inatas epiteliais e com um patógeno são melhor investigada in vivo, onde o comportamento complexo se desenrola ao longo do tempo e do espaço. No entanto, os modelos existentes não permitem fácil imaging espaço-temporal da batalha com patógenos ao nível da mucosa. O modelo desenvolvido aqui cria uma infecção das mucosas por injecção directa do agente patogénico fúngico, Candida albicans, em swimbladder de peixe-zebra juvenil. A infecção resultante permite imagens de alta resolução do comportamento das células epiteliais e imune inata ao longo do desenvolvimento da doença mucosa. A versatilidade deste método permite interrogatório do hospedeiro para sondar a seqüência detalhada de eventos imunológicos que levam a phrecrutamento agocyte e examinar os papéis de determinados tipos de células e vias moleculares na proteção. Além disso, o comportamento do agente patogénico como uma função do ataque do sistema imunológico pode ser iluminados simultaneamente por fluorescência utilizando-expressando proteína C. albicans. O aumento da resolução espacial da interação patógeno-hospedeiro também é possível através da técnica de dissecção da bexiga natatória rápida descrito. O modelo de infecção da mucosa descrito aqui é simples e altamente reprodutível, tornando-se uma ferramenta valiosa para o estudo da mucosa candidíase. Este sistema pode também ser amplamente traduzível a outros agentes patogénicos das mucosas, tais como micróbios micobacterianas, bacterianas ou virais que normalmente infectam através de superfícies epiteliais.

Microinjection na bexiga natatória posterior O método experimental aqui apresentado descreve a injecção de uma dose consistente de C. células de levedura albicans na bexiga natatória de 4 dpf peixe-zebra. Trabalhos anteriores com o modelo de imersão sugere que a resposta imune a bexiga natatória C. albicans é semelhante à da mucosa de mamífero candidíase 32. Aqui demonstramos um método de infecção modificado que é mais simples, mais rápida ...

Watch this Scientific Journal Video about Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection at JoVE.com

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Modelagem da mucosa candidíase em larval Zebrafish por swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

modeling-mucosal-candidiasis-larval-zebrafish-swimbladder

Research

JoVE Journal

Immunology and Infection

10.1K visualizações.  University of Maine. In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Vídeo: Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Não-invasivos de imagem de candidíase disseminada em larvas do peixe

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Imunologia e Infecção

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

In vitro Ensaio de cocultura para avaliar migração trans epitelial de neutrófilo induzida por patógenos

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of causing systemic disease in both fish and humans. A better understanding of S. iniae disease pathogenesis requires an appropriate model system. The genetic tractability and the optical transparency of the early developmental stages of zebrafish allow for the generation and non-invasive imaging of transgenic lines with fluorescently tagged immune cells. The adaptive immune system is not fully functional until several weeks post fertilization, but zebrafish larvae have a conserved vertebrate innate immune system with both neutrophils and macrophages. Thus, the generation of a larval infection model allows the study of the specific contribution of innate immunity in controlling S. iniae infection.
The site of microinjection will determine whether an infection is systemic or initially localized. Here, we present our protocols for otic vesicle injection of zebrafish aged 2-3 days post fertilization as well as our techniques for fluorescent confocal imaging of infection. A localized infection site allows observation of initial microbe invasion, recruitment of host cells and dissemination of infection. Our findings using the zebrafish larval model of S. iniae infection indicate that zebrafish can be used to examine the differing contributions of host neutrophils and macrophages in localized bacterial infections. In addition, we describe how photolabeling of immune cells can be used to track individual host cell fate during the course of infection.

Non-invasive Imaging of the Innate Immune Response in a Zebrafish Larval Model of Streptococcus iniae Infection

Here, we present a protocol for the generation and imaging of a localized bacterial infection in the zebrafish otic vesicle.

Imagem não invasivo do imunitário inatas Response em um modelo de peixe-zebra larval de<em&gt; Streptococcus iniae</em&gt; Infection

The aquatic pathogen, Streptococcus iniae, is responsible for over 100 million dollars in annual losses for the aquaculture industry and is capable of ...

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the analysis of immune mediators in fluid eluates for the study of the airway topical immune signature, without the need for stimulation procedures (often used by other techniques). The mucosal lining fluid is sampled on a strip of filter paper placed at the anterior part of the inferior turbinate and left for 2 min of absorption. Analytes are eluted from the filter papers, and the extracted protein-based eluates are analyzed by an electrochemiluminescence-based immunoassay, allowing for the high-sensitivity quantification of low- and high-level analytes in the same sample. We measured the in vivo levels of 20 preselected immune mediators related to specific immune signaling pathways in the upper airway mucosa, but the technique is not limited to that specific panel or sampling site. The technique was first implemented in 7-year-old children from the Copenhagen Prospective Studies on Asthma in Childhood2000 (COPSAC2000) cohort with allergic rhinitis. It was thereafter used in the longitudinal COPSAC2010 birth cohort, sampled at 1 month, 2 years, and 6 years of age and at instances of acute respiratory symptoms. We successfully obtained and analyzed samples from 620 (89%) of 700 1-month-old children; a few samples were below the assay detection limit (reported as the median (Inter-Quartile Range (IQR)). The number of samples below the detection limit (i.e.&#160;from 0 to the set point for the lower limit of detection) for each mediator was 29 (7.25 - 119.5). This technique enables the quantification of the in vivo airway mucosal immune profile from birth, can be applied longitudinally, and can be applied to studies on the effect of genetics and early-life environmental exposures, pathophysiology, endotyping, and monitoring of respiratory diseases, and development and evaluation of novel therapeutics.

Noninvasive Sampling of Mucosal Lining Fluid for the Quantification of In Vivo Upper Airway Immune-mediator Levels

This protocol describes a noninvasive technique for the sampling of undisturbed mucosal lining fluid from the upper airways. It can be used to perform the quantification of in vivo levels of protein mediators, such as cytokines and chemokines, in subjects of all ages.

Amostragem não-invasiva de mucosa líquido para a quantificação dos níveis de imune-mediador na Vivo das vias aéreas superiores

This protocol describes noninvasive sampling of undisturbed upper airway mucosal lining fluid. It also details the extraction procedure used prior to the ...

Bile Salt-induced Biofilm Formation in Enteric Pathogens: Techniques for Identification and Quantification

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, a significant stress response is induced during gastrointestinal transit and upon bile exposure, a normal component of human digestion. To overcome the bactericidal effects of bile, many enteric pathogens form a biofilm hypothesized to permit survival when transiting through the small intestine. Here we present methodologies to define biofilm formation through solid-phase adherence assays as well as extracellular polymeric substance (EPS) matrix detection and visualization. Furthermore, biofilm dispersion assessment is presented to mimic the analysis of events triggering release of bacteria during the infection process. Crystal violet staining is used to detect adherent bacteria in a high-throughput 96-well plate adherence assay. EPS production assessment is determined by two assays, namely microscopy staining of the EPS matrix and semi-quantitative analysis with a fluorescently-conjugated polysaccharide binding lectin. Finally, biofilm dispersion is measured through colony counts and plating. Positive data from multiple assays support the characterization of biofilms and can be utilized to identify bile salt-induced biofilm formation in other bacterial strains.

This protocol enables the reader to analyze bile salt-induced biofilm formation in enteric pathogens using a multifaceted approach to capture the dynamic nature of bacterial biofilms by assessing adherence, extracellular polymeric substance matrix formation, and dispersion.

Formação de biofilmes induzida por sais biliares em patógenos entéricos: técnicas para identificação e quantificação

Biofilm formation is a dynamic, multistage process that occurs in bacteria under harsh environmental conditions or times of stress. For enteric pathogens, ...

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this bacterium causes a wide disease spectrum in humans ranging from a sterilized infection to an actively progressing deadly disease. The most common form is the latent tuberculosis, which is asymptomatic, but has the potential to reactivate into a fulminant disease. Adult zebrafish and its natural pathogen Mycobacterium marinum have recently proven to be an applicable model to study the wide disease spectrum of tuberculosis. Importantly, spontaneous latency and reactivation as well as adaptive immune responses in the context of mycobacterial infection can be studied in this model. In this article, we describe methods for the experimental infection of adult zebrafish, the collection of internal organs for the extraction of nucleic acids for the measurement of mycobacterial loads and host immune responses by quantitative PCR. The in-house-developed, M. marinum-specific qPCR assay is more sensitive than the traditional plating methods as it also detects DNA from non-dividing, dormant or recently dead mycobacteria. As both DNA and RNA are extracted from the same individual, it is possible to study the relationships between the diseased state, and the host and pathogen gene-expression. The adult zebrafish model for tuberculosis thus presents itself as a highly applicable, non-mammalian in vivo system to study host-pathogen interactions.

Modeling Tuberculosis in Mycobacterium marinum Infected Adult Zebrafish

Here, we present a protocol to model human tuberculosis in an adult zebrafish using its natural pathogen Mycobacterium marinum. Extracted DNA and RNA from the internal organs of infected zebrafish can be used to reveal the total mycobacterial loads in the fish and the host&#39;s immune responses with qPCR.

Modelagem de tuberculose no Zebrafish adulto infectado de Mycobacterium marinum

Mycobacterium tuberculosis is currently the deadliest human pathogen causing 1.7 million deaths and 10.4 million infections every year. Exposure to this ...

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, Candida can overgrow their natural niches resulting in debilitating mucosal infections as well as life-threatening systemic infections, which are a major focus of investigation due to their associated high mortality rates. Animal models of disseminated infection exist for studying disease progression and dissecting the characteristics of Candida pathogenicity. Of these, the Galleria mellonella waxworm infection model provides a cost-effective experimental tool for high-throughput investigations of systemic virulence. Many other bacterial and eukaryotic infectious agents have been effectively studied in G. mellonella to understand pathogenicity, making it a widely accepted model system. Yet, variation in the method used to infect G. mellonella can alter phenotypic outcomes and complicate interpretation of the results. Here, we outline the benefits and drawbacks of the waxworm model to study systemic Candida pathogenesis and detail an approach to improve reproducibility. Our results highlight the range of mortality kinetics in G. mellonella and describe the variables which can modulate these kinetics. Ultimately, this method stands as an ethical, rapid, and cost-effective approach to study virulence in a model of disseminated candidiasis.

The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis

Galleria mellonella serves as an invertebrate model for disseminated candidiasis. Here, we detail the infection protocol and provide supporting data for the model&#39;s effectiveness.

O modelo de infecção Waxworm Galleria mellonella para candidíase disseminada

Candida species are common fungal commensals of humans colonizing the skin, mucosal surfaces, and gastrointestinal tract. Under certain conditions, ...

Induction of Mouse Lung Injury by Endotracheal Injection of Bleomycin

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse&#160;trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.

Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.

Indução de lesão de pulmão de camundongo por injeção endotraqueal de bleomicina

Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models ...

Targeting Drugs to Larval Zebrafish Macrophages by Injecting Drug-Loaded Liposomes

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells to inflammatory disease due to their functionally conserved innate immune system. They are also widely used to examine how innate immune cells help guide developmental processes. By taking advantage of the optical transparency and genetic tractability of larval zebrafish, these studies often focus on live imaging approaches to functionally characterize fluorescently marked macrophages and neutrophils within intact animals. Due to their diverse functional heterogeneity and ever-expanding roles in disease pathogenesis, macrophages have received significant attention. In addition to genetic manipulations, chemical interventions are now routinely used to manipulate and examine macrophage behavior in larval zebrafish. Delivery of these drugs is typically limited to passive targeting of free drug through direct immersion or microinjection. These approaches rely on the assumption that any changes to macrophage behavior are the result of a direct effect of the drug on the macrophages themselves, and not a downstream consequence of a direct effect on another cell type. Here, we present our protocols for targeting drugs specifically to larval zebrafish macrophages by microinjecting drug-loaded fluorescent liposomes. We reveal that poloxamer 188-modified drug-loaded blue fluorescent liposomes are readily taken up by macrophages, and not by neutrophils. We also provide evidence that drugs delivered in this way can impact macrophage activity in a manner consistent with the mechanism of action of the drug. This technique will be of value to researchers wanting to ensure targeting of drugs to macrophages and when drugs are too toxic to be delivered by traditional methods like immersion.

Here, we describe the synthesis of drug-loaded liposomes and their microinjection into larval zebrafish for the purpose of targeting drug delivery to macrophage-lineage cells.

Direcionando drogas para macrófagos de zebrafish larval injetando liposós carregados de drogas

Zebrafish (Danio rerio) larvae have developed into a popular model to investigate host-pathogen interactions and the contribution of innate immune cells ...

Isolation of Tonsillar Mononuclear Cells to Study Ex Vivo Innate Immune Responses in a Human Mucosal Lymphoid Tissue

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal immunity, because they can model host-pathogen interactions in the tissue. While isolated cells derived from tissues were the first cell culture model, their use has been neglected because tissue can be hard to obtain. In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells (TMCs) from healthy human tonsils to study innate immune responses upon activation, mimicking viral infection in mucosal tissues. Isolation of TMCs from the tonsils is quick, because the tonsils barely have any epithelium and yield up to billions of all major immune cell types. This method allows detection of cytokine production using several techniques, including immunoassays, qPCR, microscopy, flow cytometry, etc., similar to the use of peripheral mononuclear cells (PBMCs) from blood. Furthermore, TMCs show a higher sensitivity to drug testing than PBMCs, which needs to be considered for future toxicity assays. Thus, ex vivo TMCs cultures are an easy and accessible mucosal model.

In the present protocol, we explain how to easily process and culture tonsillar mononuclear cells from healthy humans undergoing partial surgical tonsillectomy to study innate immune responses upon activation, mimicking viral infection in mucosal tissues.

Isolamento de células mononucleares tonsiláres para estudar respostas imunes inatas ex vivo em um tecido linfoide mucosal humano

Studying isolated cells from mucosa-associated lymphoid tissues (MALT) allows understanding of immune cells response in pathologies involving mucosal ...