Este trabalho é financiado pelos Institutos Canadenses de Pesquisa em Saúde, Fundação Canadense para Inovação, University of British Columbia Centre for Research Blood and University of Northern BC.

1. Preparação do Reagente <ol><li> Reagentes para ser usado durante todo o experimento deve ser preparado antes de iniciar o protocolo cirúrgico. Note-se que todas as soluções foram feitas utilizando técnica estéril. </li><li> Solução salina fisiológica (PSS) é o melhor preparado em dois componentes: solução de sal básica e solução de bicarbonato de sódio. Prepare as duas soluções em 20X de concentração e armazenar a 4 ° C. Bicarbonato de sódio a 20X é 360.0mM NaHCO 3 (84.01g/mol) e solução de sal básica em 20X concentração é 2638.0mM NaCl (58.44g/mol), 94.0mM KCl (74.56g/mol), 40.0mM CaCl 2 • 2H 2 O (147.02g/mol), e 23,4 mm MgSO 4 • 7H 2 O (246.48g/mol). As soluções devem ser preparadas de dH 2 O e produtos químicos devem ser adicionados um de cada vez e agitado até que a solução é clara antes de adicionar o produto químico próxima </li><li> Prepare 2L de PSS diluindo volumes iguais de bicarbonato de sódio e solução de sal básica e diluir para 1X concentração (por duas 2L de PSS diluir 100mL de cada solução de bicarbonato de sódio e sal básico em dH 2 O </li><li> Lugar balão volumétrico de 2L contendo PSS e PSS em 37 ° C banho-maria. Equilibrar a solução com 5% de CO 2 / gás nitrogênio equilíbrio para 1hr antes do início do procedimento cirúrgico. </li><li> Produtos químicos vasoativas (ex. fenilefrina, acetilcolina, nitroprussiato de sódio) são mais bem preparados na concentração de 1M em dH2O e armazenados em 100μl alíquotas a -20 ° C. </li></ol> 2. Preparação dos animais <ol><li> Determinar o peso do mouse e administrar a dose inicial de pentobarbital sódico a 90mg/kg por via intraperitoneal. Durante todo o processo o mouse é continuamente monitorado para avião anestesia via pinch dedo do pé e um exame cuidadoso da freqüência respiratória. Injeções adicionais de pentobarbital sódico deve ser administrado conforme a necessidade de 20mg/kg de manter plano de anestesia. O plano cirúrgico de anestesia é mantida de acordo com os regulamentos federais e institucionais, conforme descrito no protocolo de atendimento de animais aprovado pelo Comitê Animal Care e Use (ACUC) da Universidade do Norte do BC. </li><li> Remover os pêlos da região abdominal e flanco / hip pela máquina de barbear. Remover restantes cabelo solto por gaze com álcool e acariciando a área com fita adesiva. Isto é suficiente como este procedimento experimental é terminal. </li><li> Posicione o mouse em uma placa clara cirúrgica em posição supina e anexe o mouse para a placa gravando cada pata para o conselho. Temperatura corporal é mantida através de uma lâmpada de calor externo e monitorados via termômetro retal para garantir que a hipotermia não ocorre como resultado de anestésico. </li></ol> 3. Procedimento Cirúrgico <ol><li> Durante todo o procedimento cirúrgico garantir tecido exposto é sempre mantido superfused com solução equilibrada PSS aquecido. Também é importante nunca fazer o contato com a vasculatura de interesse com instrumentos cirúrgicos ou ferir os vasos, colocando a força muito sobre eles através da manipulação de tecido adiposo e tecido conjuntivo em torno deles. </li><li> Posicione o mouse no âmbito de dissecação. Faça uma incisão na linha média ao longo da superfície ventral da cavidade abdominal e retrair a pele para a coluna vertebral do rato. </li><li> Quando a pele é retirada para que a LN é facilmente visível, o pino da pele em um pedestal de Sylgard transparente 184. </li><li> Remover a fina camada de tecido conjuntivo sobrepondo a área ao redor do LN </li><li> Clara a sobreposição de tecido adiposo e cercam o nó de linfa até o leito microvascular está exposto. O principal segmento arterial estabelece adjacente ao LN e é comumente sobrepostos pela vênula. Toda a cirurgia demora cerca de 30 minutos. </li></ol> 4. Análise de imagem e navio <ol><li> Uma vez que o segmento de interesse navio está exposta no âmbito de dissecação, assegurar a preparação no microscópio intravital. Note-se que animal permanece no conselho claro cirúrgica, enquanto no microscópio intravital durante toda a duração do experimento. A temperatura corporal é mantida através de lâmpada de calor e medido através de termômetro retal, conforme descrito durante a seção cirúrgica. O avião anestesia também é monitorada durante todo o experimento como descrito na seção 2.1. </li><li> Configure o superfusão e linhas de sucção. Superfusão do PSS ocorre por alimentação por gravidade a partir de um reservatório, no reservatório jaqueta de água aquecida (a jaqueta de água é distribuída e aquecida pelo banho de água circulante) de perfundidos por 5% de nitrogênio CO 2 equilibrado, e para baixo uma linha de gotejamento a uma taxa de 10ml / min. A linha de sucção deve ser usado para continuar a puxar PSS superfused longe da preparação. Note-se que antes do experimento as linhas de sucção e uma jaqueta de água são cuidadosamente limpos e armazenados após cada experimento. Isso garante a esterilidade antes do próximo experimento. </li><li> Verifique a temperatura do PSS superfusing. Ajustar a temperatura docirculando banho de água e / ou a taxa de superfusão PSS para atingir uma temperatura de ~ 37 ° C em toda a preparação. </li><li> Permitir a vasculatura para equilibrar com PSS para pelo menos 60 minutos e quantificar o diâmetro do vaso usando o paquímetro de vídeo. Tipicamente, o diâmetro dos vasos deve ser determinada em vários pontos (por exemplo, em 40, 50 e 60 minutos) para garantir que o navio tenha estabilizado. </li><li> Avaliar a saúde do navio usando um agonista do músculo liso específica (ex. Fenilefrina, PE) e um agonista endotélio específico (ex. acetilcolina, ACh). Concentração de agonista deve ser ajustada dependendo do agonista, mas para PE e Ach o navio deve ser avaliado em cada concentração pela adição cumulativa (10-5M a 10-9M) para o superfusate. Cada aplicação agonista devem ser separados por uma fase de lavagem (normalmente 30 minutos) usando PSS, até o navio retorna ao diâmetro de descanso. </li><li> Após a avaliação da saúde dos vasos, após uso de drogas vasoativas, rotulagem fluorescentes e rastreamento de células experimentos podem ser realizados. </li><li> Após o final do experimento o animal recebe uma overdose de pentobarbital sódico. O animal é então monitorado para garantir que não há resposta para beliscar dedo do pé e falta de movimento respiratório ou batimentos cardíacos. Nesse momento esse é seguido por deslocamento cervical. </li></ol> 5. Resultados representativos: Após o período de equilíbrio a preparação deve resultar em uma imagem clara que permite a identificação de ambos os arteríola e vênula que abastece o nó de linfa inguinal. Deve haver células adiposas mínima em torno dos vasos para permitir paredes da arteríola e vênula ser claramente observado para as pinças de vídeo para ser sobreposta para calcular o diâmetro dos vasos. O ponto de integrante de uma preparação bem sucedida é a arteríola tem um rico suprimento de sangue em movimento através da luz e que há um grau significativo de tom vascular (isto é, a arteríola tem a capacidade de vasoconstrição e dilatam de músculo liso e endoteliais agonistas específicos, respectivamente) . <img src="/files/ftp_upload/2551/2551fig1.jpg" alt="Figura 1"/> Figura 1. Imagem de microscopia intravital de linfonodo inguinal alimentar arteríola (seta vermelha) alimentando o linfonodo equilibrada com solução salina fisiológica e correr ao lado do principal vênula (seta verde). <img src="/files/ftp_upload/2551/2551fig2.jpg" alt="Figura 2"/> Figura 2. Vasoativas resposta normal à fenilefrina superfused (PE) pela adição cumulativa de soro fisiológico a partir das concentrações 10 M -9 a 10 -5 M. A presença de uma vasoconstrição robusta é sugestivo de função muscular normal liso presente na arteríola. <img src="/files/ftp_upload/2551/2551fig3.jpg" alt="Figura 3"/> Figura 3. Vasoativas resposta normal à acetilcolina superfused (ACh) pela adição cumulativa de soro fisiológico das concentrações de 10 M -9 a 10 -5 M. A presença de uma vasodilatação robusta é sugestiva de apresentar função normal endotelial na arteríola.

<ol>
	<li>Bearden, S. E., Payne, G. W., Chisty, A., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arteriolar+network+architecture+and+vasomotor+function+with+ageing+in+mouse+gluteus+maximus+muscle.">Arteriolar network architecture and vasomotor function with ageing in mouse gluteus maximus muscle.</a> J Physiol. 561, 535-545 (2004).</li><li>Looft-Wilson, R. C., Payne, G. W., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connexin+expression+and+conducted+vasodilation+along+arteriolar+endothelium+in+mouse+skeletal+muscle.">Connexin expression and conducted vasodilation along arteriolar endothelium in mouse skeletal muscle.</a> J Appl Physiol. 97, 1152-1158 (2004).</li><li>Payne, G. W., Madri, J. A., Sessa, W. C., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histamine+inhibits+conducted+vasodilation+through+endothelium-derived+NO+production+in+arterioles+of+mouse+skeletal+muscle.">Histamine inhibits conducted vasodilation through endothelium-derived NO production in arterioles of mouse skeletal muscle.</a> Faseb J. 18, 280-286 (2004).</li><li>Smeda, J. S., Payne, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+autoregulatory+and+myogenic+function+in+the+cerebrovasculature+of+Dahl+salt-sensitive+rats.">Alterations in autoregulatory and myogenic function in the cerebrovasculature of Dahl salt-sensitive rats.</a> Stroke. 34, 1484-1490 (2003).</li><li>de With, M. C., de Vries, A. M., Kroese, A. B., van der Heijden, E. P., Bleys, R. L., Segal, S. S., Kon, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+anatomy+of+the+hamster+retractor+muscle+with+regard+to+its+microvascular+transfer.">Vascular anatomy of the hamster retractor muscle with regard to its microvascular transfer.</a> Eur Surg Res. 42, 97-105 (2009).</li><li>Domeier, T. L., Segal, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electromechanical+and+pharmacomechanical+signalling+pathways+for+conducted+vasodilatation+along+endothelium+of+hamster+feed+arteries.">Electromechanical and pharmacomechanical signalling pathways for conducted vasodilatation along endothelium of hamster feed arteries.</a> J Physiol. 579, 175-186 (2007).</li></ol>

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Microscopia intravital do linfonodo inguinal apresentado aqui fornece a capacidade de microvasculatura imagem do linfonodo in-vivo. Assim, facilita um meio de direta, a observação em tempo real. Imagem da microvasculatura LN é um site exclusivo que permite estudar a interface entre a resposta imune e da vasculatura. Usando essa preparação, o foco pode ser direcionado especificamente a resposta imune, alteração da vasculatura, ou a interação entre os dois. Tal como acontece com todas as abordagens experimentais, microscopia intravital padrão tem vantagens e limitações. IVM padrão, tais como a preparação descrito aqui, pode ser facilmente modificado, e tem sido demonstrado anteriormente pelos autores para permitir que a microscopia de epifluorescência através da introdução de corantes traçador fluorescente ou populações de células marcadas. Embora IVM norma não dá a possibilidade de três dimensional imagem e rastreamento, como seria dada por dois fótons de microscopia ou angiografia, rastreamento de celular ainda é realizado em duas dimensões permitindo célula a célula e interação célula-vasculatura a ser observados e quantificados e em conjunto com in-vivo administração e posterior coloração com anticorpos fluorescentes podem ser usados ​​para dar dados sobre proteína / marcador de expressão em tempo real. Nomeadamente, técnicas de imagem alternativas exigem um ambiente de estática para imagens; fixação da área cirúrgica ou a adição de uma lamela em um flat de preparação é frequentemente necessário. Isso limita, se não elimina, a capacidade de ativamente perfundir mediadores vascular ou outros sobre a preparação para avaliar a integridade vascular e fisiologia etc, ou o uso de outras técnicas, tais como vasodilatação experimentos realizados no preparo IVM. Além disso, o padrão IVM não exige uma preparação totalmente plana e não é afetado pelo movimento, como o gerado pela respiração do animal. Isto permite IVM padrão a ser usado em mais áreas cirúrgicas com maior reprodutibilidade. Isto é exemplificado pela preparação de linfonodo inguinal descrita aqui. Dado o tamanho ea forma do nó de linfa, a preparação não pode ser feita plana sem ferir o tecido ea localização do nó dá origem ao movimento significativo devido à respiração animal. Questões como seria difícil de superar por outros métodos, mas são facilmente tratada com a preparação IVM padrão descrito. Em resumo, a preparação detalhada acima podem ser combinadas com qualquer número de outras bioquímicas, vasculares e / ou técnicas imunológicas, tais como transferência de subconjuntos de ativado ou células marcadas, a indução de hipóxia, e sobre-expressão do esgotamento de mediadores vasculares. No entanto, as aplicações adicionais são dependentes da saúde da preparação inicial. Portanto, é vital para sempre testar a saúde da vasculatura sendo fotografada e avaliada. Pontos chave para conseguir uma preparação de saúde estão garantindo a preparação é constante perfundidos com PSS equilibrada a temperatura corporal e minimizando o contato e estresse colocado na área cirúrgica durante a cirurgia.

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th></tr><tr><td> Bicarbonato de sódio </td><td> Sigma Aldrich </td></tr><tr><td> Cloreto de sódio </td><td> Sigma Aldrich </td></tr><tr><td> Cloreto de Potássio </td><td> Sigma Aldrich </td></tr><tr><td> Cloreto de cálcio dihidratado </td><td> Sigma Aldrich </td></tr><tr><td> Nitroprussiato de sódio </td><td> Sigma Aldrich </td></tr><tr><td> Fenilefrina </td><td> Sigma Aldrich </td></tr><tr><td> Acetilcolina </td><td> Sigma Aldrich </td></tr><tr><td> Cloreto de magnésio hepta-hidratado </td><td> Sigma Aldrich </td></tr><tr><td> Sódio Pentobarbitol </td><td></td></tr></tbody></table>

intravital microscopy of the inguinal lymph node

Linfonodos (LN é), localizados em todo o corpo, são um componente integral do sistema imunológico. Eles servem como um local para indução de resposta imune adaptativa e, portanto, o desenvolvimento de células efetoras. Como tal, linfonodos são fundamentais para combater patógenos invasores e manter a saúde. A escolha do LN para estudar é ditada pela acessibilidade e o modelo desejado; o linfonodo inguinal é bem localizada e de fácil apóia estudos de modelos biologicamente relevantes de pele e infecção da mucosa genital. A LN inguinal, como todos os linfonodos, tem uma rede microvascular extensa fornecê-lo com sangue. Em geral, esta rede microvascular inclui a arteríola alimentação principal da LN que, posteriormente, ramos e alimenta alta vênulas endoteliais (VEH). VHE são especializados para facilitar o tráfico de células imunes para o LN durante tanto homeostase e infecção. Como HEVs regulam o tráfico no LN em ambas as circunstâncias é uma área de intensa exploração. A LN alimentar arteríola, tem influência direta sobre o montante HEVs e é a principal fonte de nutrientes e sangue rico em células do LN. Além disso, as mudanças na arteríola alimentar estão implicados no sentido de facilitar a indução da resposta imune adaptativa. A microvasculatura LN tem uma importância óbvia para manter um suprimento de sangue ótima para o LN e regular fluxo de células imunes para o LN, que são elementos cruciais na função LN adequada e resposta imune posteriormente. A capacidade de estudo da microvasculatura LN in vivo é a chave para elucidar como o sistema imunológico e da microvasculatura interagem e influenciam-se mutuamente dentro da LN. Aqui, apresentamos um método in vivo para a imagem do linfonodo inguinal. Nós nos concentramos em imagens da microvasculatura da LN, prestando particular atenção aos métodos que garantem o estudo dos vasos saudáveis, a capacidade de manter imagens de vasos viáveis ​​ao longo de um número de horas, e quantificação da magnitude do vaso. Métodos para a perfusão da microvasculatura com drogas vasoativas, bem como o potencial para rastrear e quantificar o tráfego celular também são apresentados. Microscopia intravital da LN inguinal permite avaliação direta da funcionalidade microvascular e em tempo real da interface direta entre células do sistema imunológico, o LN, e da microcirculação. Esta técnica potencial para ser combinada com técnicas imunológicas muitos e rotulagem de células fluorescentes, bem como manipulado para estudar vasculatura de linfonodos outros.

Watch this Scientific Journal Video about Intravital Microscopy of the Inguinal Lymph Node at JoVE.com

Intravital Microscopy of the Inguinal Lymph Node

Uma técnica para a realização de microscopia intravital do linfonodo inguinal (LN) é descrito. Tal técnica permite em tempo real,<em&gt; In vivo</em&gt; Estudo da microvasculatura linfonodo e estrutura, tanto durante a homeostase e infecção. Esta técnica pode ser adaptada para estudos de células e tráfico para sites outro nó de linfa.

Microscopia intravital do linfonodo inguinal

Lymph nodes (LN's), located throughout the body, are an integral component of the immune system. They serve as a site for induction of adaptive immune ...

intravital-microscopy-of-the-inguinal-lymph-node

Research

JoVE Journal

Immunology and Infection

19.9K visualizações.  University of Northern British Columbia. Uma técnica para a realização de microscopia intravital do linfonodo inguinal (LN) é descrito. Tal técnica permite em tempo real, In vivo Estudo da microvasculatura linfonodo e estrutura, tanto durante a homeostase e infecção. Esta técnica pode ser adaptada para estudos de células e tráfico para sites outro nó de linfa.

Vídeo: Intravital Microscopy of the Inguinal Lymph Node

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Ex vivo de imagens de células T em fatias Node murino linfáticos com Widefield e microscópios confocal

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

Imunologia e Infecção

Murine Superficial Lymph Node Surgery

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often followed over time. Similarly, the study of lymphocyte homeostasis requires time course experiments. Performing these studies within the same mouse is ideal to reduce the experimental variability as well as the number of mice used. Blood withdrawal allows performance of time course experiments, but it only gives information about circulating lymphocytes and provides a limited number of cells1-4. Since lymphocytes circulating through the body and residing in the lymph nodes have different properties, it is important to examine both locations. The sequential removal of lymph nodes by surgery provides a unique opportunity to follow an immune response or immune cell expansion in the same mouse over time. Furthermore, this technique yields between 1-2x106 cells per lymph node which is sufficient to perform phenotypic characterization and/or functional assays. Sequential lymph node surgery or lymphadenectomy has been successfully used by us and others5-11. Here, we describe how the brachial and inguinal lymph nodes can be removed by making a small incision in the skin of an anesthetised mouse. Since the surgery is superficial and done rapidly, the mouse recovers very quickly, heals well and does not experience excessive pain. Every second day, it is possible to harvest one or two lymph nodes allowing for time course experiments. This technique is thus suitable to study the characteristics of lymph node-residing lymphocytes over time. This approach is suitable to various experimental designs and we believe that many laboratories would benefit from performing sequential lymph node surgeries.

To follow the progression of an immune response over time within the same mouse, lymph nodes can be sequentially removed by surgery. Here, we describe how this technique can be performed.

Cirurgia Nó murino superficial linfático

In the field of immunology, to understand the progression of an immune response against a vaccine, an infection or a tumour, the response is often ...

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic tools and surgical procedures, TPM becomes a powerful technique to identify "niches" inside organs and to document cellular "behaviors" in live animals. While intravital imaging provides information that best resembles the real cellular behavior inside the organ, it is both more laborious and technically demanding in terms of required equipment/procedures than alternative ex vivo imaging acquisition. Thus, we describe a surgical procedure and novel "stereotactic" organ holder that allows us to follow the movements of Foxp3+ cells within the thymus.
Foxp3 is the master regulator for the generation of regulatory T cells (Tregs). Moreover, these cells can be classified according to their origin: ie. thymus-differentiated Tregs are called "naturally-occurring Tregs" (nTregs), as opposed to peripherally-converted Tregs (pTregs). Although significant amount of research has been reported in the literature concerning the phenotype and physiology of these T cells, very little is known about their in vivo interactions with other cells. This deficiency may be due to the absence of techniques that would permit such observations. The protocol described in this paper provides a remedy for this situation.
Our protocol consists of using nude mice that lack an endogenous thymus since they have a punctual mutation in the DNA sequence that compromises the differentiation of some epithelial cells, including thymic epithelial cells. Nude mice were gamma-irradiated and reconstituted with bone marrows (BM) from Foxp3-KIgfp/gfp mice. After BM recovery (6 weeks), each animal received embryonic thymus transplantation inside the kidney capsule. After thymus acceptance (6 weeks), the animals were anesthetized; the kidney containing the transplanted thymus was exposed, fixed in our organ holder, and kept under physiological conditions for in vivo imaging by TPM. We have been using this approach to study the influence of drugs in the generation of regulatory T cells.

We have developed novel laboratory tools and protocols for intravital imaging acquisition of the thymus. Our technique should help in the identification of &#x201C;niches&#x201D; within the thymus where T cell development occurs.

Imagem intravital da Thymus rato usando 2-Photon Microscopia

Two-photon Microscopy (TPM) provides image acquisition in deep areas inside tissues and organs. In combination with the development of new stereotactic ...

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

Microscopia intravital do Baço: Análise Quantitativa da Mobilidade Parasite e do fluxo sanguíneo

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

Intravital Imaging of the Mouse Popliteal Lymph Node

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign antigens from peripheral tissues to prime the adaptive immune response. Juxtaposed between innate and adaptive immune responses, the LN is an ideal site to study immune cell interactions1,2. Lymphocytes (T cells, B cells and NK cells), dendritic cells (DCs), and macrophages comprise the bulk of bone marrow-derived cellular elements of the LN. These cells are strategically positioned in the LN to allow efficient surveillance of self antigens and potential foreign antigens3-5. The process by which lymphocytes successfully encounter cognate antigens is a subject of intense investigation in recent years, and involves an integration of molecular contacts including antigen receptors, adhesion molecules, chemokines, and stromal structures such as the fibro-reticular network2,6-12. 
Prior to the development of high-resolution real-time fluorescent in vivo imaging, investigators relied on static imaging, which only offers answers regarding morphology, position, and architecture. While these questions are fundamental in our understanding of immune cell behavior, the limitations intrinsic with this technique does not permit analysis to decipher lymphocyte trafficking and environmental clues that affect dynamic cell behavior. Recently, the development of intravital two-photon laser scanning microscopy (2P-LSM) has allowed investigators to view the dynamic movements and interactions of individual cells within live LNs in situ12-16. In particular, we and others have applied this technique to image cellular behavior and interactions within the popliteal LN, where its compact, dense nature offers the advantage of multiplex data acquisition over a large tissue area with diverse tissue sub-structures11,17-18. It is important to note that this technique offers added benefits over explanted tissue imaging techniques, which require disruption of blood, lymph flow, and ultimately the cellular dynamics of the system. Additionally, explanted tissues have a very limited window of time in which the tissue remains viable for imaging after explant. With proper hydration and monitoring of the animal's environmental conditions, the imaging time can be significantly extended with this intravital technique. Here, we present a detailed method of preparing mouse popliteal LN for the purpose of performing intravital imaging.

Recent advances in 2-photon microscopy have enabled real-time in situ imaging of live tissues in animal models, thereby enhancing our ability to investigate cellular behavior in both physiologic and pathologic conditions. Here, we outline the preparations required to perform intravital imaging of the mouse popliteal lymph node.

Imagem intravital do Linfonodo Popliteal Rato

Lymph nodes (LNs) are secondary lymphoid organs, which are strategically located throughout the body to allow for trapping and presentation of foreign ...

Generation of Lymph Node-fat Pad Chimeras for the Study of Lymph Node Stromal Cell Origin

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the mechanisms governing its formation. Lymph nodes are always encapsulated in adipose tissue and we recently demonstrated the importance of this relation for the formation of lymph node stroma. Adipocyte precursor cells migrate into the lymph node during its development and upon engagement of the Lymphotoxin-b receptor switch off adipogenesis and differentiate into lymphoid stromal cells (B&#233;n&#233;zech&#160;et al.14). Based on the lymphoid stroma potential of adipose tissue, we present a method using a lymph node/fat pad chimera that allows the lineage tracing of lymph node stromal cell precursors. We show how to isolate newborn lymph nodes and EYFP+ embryonic adipose tissue and make a LN/ EYFP+ fat pad chimera. After transfer under the kidney capsule of a host mouse, the lymph node incorporates local adipose tissue precursor cells and finishes its formation. Progeny analysis of EYFP+ fat pad cells in the resulting lymph nodes can be performed by flow-cytometric analysis of enzymatically digested lymph nodes or by immunofluorescence analysis of lymph nodes cryosections. By using fat pads from different knockout mouse models, this method will provide an efficient way of analyzing the origin of the different lymph node stromal cell populations.

Generation of lymph node/fat pad chimeras for the study of lymph node stromal cell origin is described. The method involves the isolation of lymph nodes from newborn mice and embryonic fat pads, the generation of chimeric lymph node-fat pads, and their transfer under the kidney capsule of a host mouse.

The stroma is a key component of the lymph node structure and function. However, little is known about its origin, exact cellular composition and the ...

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick tissue samples and organs in health and disease. By controlling the scanning pattern in multi-photon microscopy and applying appropriate numerical algorithms, we developed a striped-illumination approach, which enabled us to achieve 3-fold better axial resolution and improved signal-to-noise ratio, i.e. contrast, in more than 100 &#181;m tissue depth within highly scattering tissue of lymphoid organs as compared to standard multi-photon microscopy. The acquisition speed as well as photobleaching and photodamage effects were similar to standard photo-multiplier-based technique, whereas the imaging depth was slightly lower due to the use of field detectors. By using the striped-illumination approach, we are able to observe the dynamics of immune complex deposits on secondary follicular dendritic cells &#8211; on the level of a few protein molecules in germinal centers.

High-resolution intravital imaging with enhanced contrast up to 120 &#181;m depth in lymph nodes of adult mice is achieved by spatially modulating the excitation pattern of a multi-focal two-photon microscope. In 100 &#181;m depth we measured resolutions of 487 nm (lateral) and 551 nm (axial), thus circumventing scattering and diffraction limits.

Altamente Resolvido Microscopia intravital Striped-iluminação dos centros germinativos

Monitoring cellular communication by intravital deep-tissue multi-photon microscopy is the key for understanding the fate of immune cells within thick ...

Isolation of Murine Lymph Node Stromal Cells

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and differentiation of lymphocytes. Various stromal cell subsets have been identified by the expression of the adhesion molecule CD31 and glycoprotein podoplanin (gp38), T zone reticular cells or fibroblastic reticular cells, lymphatic endothelial cells, blood endothelial cells and FRC-like pericytes within the double negative cell population. For all populations different functions are described including, separation and lining of different compartments, attraction of and interaction with different cell types, filtration of the draining fluidics and contraction of the lymphatic vessels. In the last years, different groups have described an additional role of stromal cells in orchestrating and regulating cytotoxic T cell responses potentially dangerous for the host. 
Lymph nodes are complex structures with many different cell types and therefore require a appropriate procedure for isolation of the desired cell populations. Currently, protocols for the isolation of lymph node stromal cells rely on enzymatic digestion with varying incubation times; however, stromal cells and their surface molecules are sensitive to these enzymes, which results in loss of surface marker expression and cell death. Here a short enzymatic digestion protocol combined with automated mechanical disruption to obtain viable single cells suspension of lymph node stromal cells maintaining their surface molecule expression is proposed.

Isolation of lymph node stromal cells is a multistep procedure including enzymatic digestion and mechanical disaggregation to obtain fibroblastic reticular cells, lymphatic and blood endothelial cells. In the described procedure, a short digestion is combined with automated mechanical disaggregation to minimize surface marker degradation of viable lymph node stromal cells.

Isolamento de Murino Linfonodo células estromais

Secondary lymphoid organs including lymph nodes are composed of stromal cells that provide a structural environment for homeostasis, activation and ...

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various biological events in real time and in live animals. This technology has greatly advanced our understanding of physiological processes and pathogen-mediated phenomena in specific organs.
In this study, IVM is applied to the mouse liver and protocols are designed to image in vivo the circulatory system of the liver and measure red blood cell (RBC) velocity in individual hepatic vessels. To visualize the different vessel subtypes that characterize the hepatic organ and perform blood flow speed measurements, C57Bl/6 mice are intravenously injected with a fluorescent plasma reagent that labels the liver-associated vasculature. IVM enables in vivo, real time, measurement of RBC velocity in a specific vessel of interest. Establishing this methodology will make it possible to investigate liver hemodynamics under physiological and pathological conditions. Ultimately, this imaging-based methodology will be important for studying the influence of L. donovani infection&#160;on hepatic hemodynamics.
This method can be applied to other infectious models and mouse organs and might be further extended to pre-clinical testing of a drug&#8217;s effect on inflammation by quantifying its effect on blood flow.

Intravital Microscopy Imaging of the Liver following Leishmania Infection: An Assessment of Hepatic Hemodynamics

This article reports on a detailed method for the dynamic measurement and quantification of blood flow velocity within individual blood vessels of the mouse liver vasculature using intravital microscopy imaging in combination with a specific methodology for image acquisition and analysis.

Microscopia intravital Imagem do Fígado seguinte<em&gt; Leishmania</em&gt; Infecção: Uma Avaliação de hepáticas Hemodinâmica

Intravital microscopy (IVM) is a powerful optical imaging technique that has made possible the visualization, monitoring and quantification of various ...

Collection and Processing of Lymph Nodes from Large Animals for RNA Analysis: Preparing for Lymph Node Transcriptomic Studies of Large Animal Species

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and E. coli, and are useful for the study of pathogenesis and/or spread of the bacteria in natural hosts. With the key function of lymph nodes in the host immune response, lymph node tissues serve as a potential source of RNA for downstream transcriptomic analyses, in order to assess the temporal changes in gene expression in cells over the course of an infection. This article presents an overview of the process of lymph node collection, tissue sampling, and downstream RNA processing in livestock, using cattle (Bos taurus) as a model, with additional examples provided from the American bison (Bison bison). The protocol includes information about the location, identification, and removal of lymph nodes from multiple key sites in the body. Additionally, a biopsy sampling methodology is presented that allows for a consistency of sampling across multiple animals. Several considerations for sample preservation are discussed, including the generation of RNA suitable for downstream methodologies like RNA-sequencing and RT-PCR. Due to the long delays inherent in large animal vs. mouse time course studies, representative results from bison and bovine lymph node tissues are presented to describe the time course of the degradation in this tissue type, in the context of a review of previous methodological work on RNA degradation in other tissues. Overall, this protocol will be useful to both veterinary researchers beginning transcriptome projects on large animal samples and to molecular biologists interested in learning techniques for in vivo tissue sampling and in vitro processing.

This protocol provides an overview of procedures for the isolation of RNA for the transcriptomic profiling of lymph node tissues from large animals, including steps in the identification and excision of lymph nodes from livestock and wildlife, sampling approaches to provide consistency across multiple animals, and considerations plus representative results for the post-collection preservation and processing for RNA analysis.

Coleta e processamento de linfonodos de animais de grandes porte para a análise do RNA: preparando-se para estudos de linfonodo Transcriptomic de espécies de animais grandes

Large animals (both livestock and wildlife) serve as important reservoirs of zoonotic pathogens, including Brucella, Mycobacterium bovis, Salmonella, and ...