We wish to thank Rebecca Paquette for assistance with reagent preparation. MPM is supported by a Ruth L. Kirchstein predoctoral fellowship from the National Institutes of Health and support from grants GM-41514 (MDC), NS-48252 (KGC), GM-48071 (IP) also from the National Institutes of Health.

1. Adoptive transfer of cells:
Tail vein or intraocular injection are both suitable methods for the introduction of cell tracker-labeled or fluorescent protein-expressing lymphocytes. In this video. we demonstrate adoptive transfer of cells by tail vein injection.
a. Materials
CD4+ T cells are labeled with 1.4 &mu;M CFSE for 30 minutes at 37&ordm;C, washed twice, and re-suspended in 100 &mu;L of RPMI-1640. Cells are loaded into a sterile 28 gauge insulin syringe for injection. The number of cells used depends on the experiment being done. Typically, 5 million T cells&nbsp;are enough for simple T cell visualization. A rodent restrainer for the mouse is needed to prevent injury to the animal&nbsp;and to&nbsp;yourself.
b. Securing the animal
The mouse is secured for adoptive transfer by slowly backing the animal into the rodent restrainer (see video) and closing the open end of the tube by gently depressing the plunger. Do not let go of the mouse tail during this process, as some smaller animals can turn around inside the restrainer. This method prevents the animal from injuring itself and from moving during the injection. When setting up the rodent restrainer, do not push the plunger in further than is necessary to keep the animal from jumping forward. Also,&nbsp;do not push the plunger against the animal, and do not leave the animal in the restrainer for an extended period of time.
c. Visualizing the tail vein
Gently pull the tail towards you so that it is lightly wrapped over your index finger and held against the inside of the finger by your thumb. Identify the tail veins located on either side of the upright tail. Visualization is facilitated by warming the tail with warm water, then wiping down with ethanol.
d. Injection of cells
When the vein has been located, insert the syringe bevel-side up at a shallow angle parallel to the vein. Once in the vein, gently depress the plunger; if you are in the vein there should be NO resistance to the injection. If there is ANY resistance, remove the syringe and try again in a different location.
If you are in the vein, slowly depress the plunger until the injection is complete. The tail should not become distended during the injection. If it does, this would mean that the needle is not in the vein. It is possible to "lose" the vein during the injection. If this happens, remove the needle and try your injection in a different spot. If you need to attempt more than one injection, it is best to move the tail up towards the body of the mouse from the original injection site.&nbsp;Plan ahead for this possibility by starting distally on the tail to give yourself room for another attempt at the adoptive transfer.
e. Post-injection considerations
After the injection is completed, a small back-flow of blood from the vein will occur. Gently press the injection site with a clean-wipe until the bleeding stops. Remove the plunger and let the animal walk forward, out of the restrainer, while gently holding onto the tail.
Always check with your animal care committee and animal use protocols to ensure you are in compliance. It is important to practice this technique several times before trying the real experiment.
2. Lymph Node Isolation
Isolation of lymph nodes for two photon imaging requires careful removal of the correct lymph node from the surrounding tissue.
a. Selection and location of peripheral lymph nodes
Selection of lymph nodes for imaging will depend on the experimental goals. Be aware of local infection or injection-draining lymph nodes, and select the appropriate node for imaging. The article by Van den Broeck et al., describes the location and morphology of murine lymph nodes in detail3. In this video, we demonstrate the excision of six large peripheral lymph nodes which are suitable for cell isolation or imaging.
b. Lymph node removal:
Keep in mind the integrity of the tissue while removing lymph nodes. Be careful not to rupture the capsule or in any way damage the lymph node structure. Loss of lymph node structural integrity will compromise the experiment. If necessary, remove fat from the lymph node surface with fine dissecting (#5 Dumont) forceps under a dissecting microscope. Lymph nodes should not have any remaining fat on the surface of the node, as this will obscure imaging by scattering the light.
Proper excision of lymph nodes is also an important technique to practice before the first real experiment.
3. Lymph Node Imaging Setup and Considerations
The lymph node should be secured to the imaging stage, which&nbsp;in this case consists of a recessed chamber. Warmed, oxygen-perfused media is pumped into the chamber from one side using a peristaltic pump and inline heater, and it exits via a downward-graded channel into a collecting chamber with a vacuum tube to a waste collection flask. In our system, the lymph node is submerged in 37&deg;C media and super-perfused with medical grade carbogen (5% CO2, 95% O2). The temperature of the perfusion media should be recorded as close to the lymph node as is possible. Your particular system may require variations in the media flow rate and stage design to accommodate the microscope stage and objective.
a. Lymph node imaging setup:
As shown in the video, we secure the lymph node by first attaching it, medullary-side down (to image T or B cell zones using an upright microscope) to a plastic coverslip, cut to the appropriate size. It is important to use a non-toxic tissue glue (we use VetBond&trade; from 3M Corporation).
The coverslip is secured in the flowing media by placing a small dab of silicone grease on the underside of the cover slip, then adhering this to the glass base of the imaging chamber.
b. 2-Photon imaging considerations:
Several factors can cause lymphocytes to stop moving: temperature that is too high or too low; excess laser power; compression or other damage to the lymph node; and lack of oxygen. Damage due to excessive laser power or temperatures &gt; ~39&deg;C is irreversible.
If cells are dim, it is typically better to increase the detector gain or the cell-labeling protocol rather than using a higher laser power to visualize cells. Optimization of labeling or expression of fluorescent proteins may be required to bring the fluorescence above the level of background autofluorescence. With reasonable gain and laser power settings, about a two-log shift of fluorescence above baseline (measured by flow cytometry) is reasonable for cell visualization.
With tunable two-photon lasers, the wavelength used for excitation should be a little less than double the single-photon excitation maximum of the fluorophore being imaged. In two-photon experiments using more than one label or fluorescent protein it may be necessary to experiment with the excitation wavelength used to find the optimal settings for both labels. Two-photon excitation can also be used to image endogenous collagen structures by taking advantage of the intrinsic blue second harmonic generation. Second harmonic generation (SHG) occurs where two photons are combined within a material and emit a single photon of twice the frequency of the original photons. In tissue, two-photon excitation occurs when the incident laser light is perpendicular to a highly ordered protein structure such as collagen.
Practical issues regarding two-photon imaging were discussed in preview reviews4, 5.

<ol>
	<li>Cahalan, M. D., Parker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choreography+of+Cell+Motility+and+Interaction+Dynamics+Imaged+by+Two-Photon+Microscopy+in+Lymphoid+Organs.+Annu+Rev+Immunol.">Choreography of Cell Motility and Interaction Dynamics Imaged by Two-Photon Microscopy in Lymphoid Organs. Annu Rev Immunol.</a> Annu Rev Immunol. , (2008).</li><li>Miller, M. J., Wei, S. H., Parker, I., Cahalan, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+imaging+of+lymphocyte+motility+and+antigen+response+in+intact+lymph+node.">Two-photon imaging of lymphocyte motility and antigen response in intact lymph node.</a> Science. 296, 1869-1873 (2002).</li><li>Van den Broeck, W., Derore, A., Simoens, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+nomenclature+of+murine+lymph+nodes:+Descriptive+study+and+nomenclatory+standardization+in+BALB/cAnNCrl+mice.">Anatomy and nomenclature of murine lymph nodes: Descriptive study and nomenclatory standardization in BALB/cAnNCrl mice.</a> J Immunol Methods. 312, 12-19 (2006).</li><li>Cahalan, M. D., Parker, I., Wei, S. H., Miller, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+tissue+imaging:+seeing+the+immune+system+in+a+fresh+light.">Two-photon tissue imaging: seeing the immune system in a fresh light.</a> Nat Rev Immunol. 2, 872-880 (2002).</li><li>Germain, R. N., Miller, M. J., Dustin, M. L., Nussenzweig, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+imaging+of+the+immune+system:+progress,+pitfalls+and+promise.">Dynamic imaging of the immune system: progress, pitfalls and promise.</a> Nat Rev Immunol. 6, 497-507 (2006).</li></ol>

In this video protocol we show the procedures for adoptive cell transfer and lymph node isolation and preparation required for imaging lymphocyte motility in a peripheral lymph node. Two-photon imaging has several advantages over confocal imaging in both explanted tissues (shown here) and in intravital preparations. Notably, use of infra-red excitation minimizes light scattering and allows for imaging ~300 micrometers beneath the capsule of the lymph node and deeper in some tissues4. Moreover, multi-photon excitation is restricted to the focal point of the objective, minimizing photo bleaching and tissue damage out side of the focal plane. As with any new technique, however, two-photon imaging is not a panacea and its limitations and potential pitfalls must be kept in mind5. Among these is the requirement that - excepting intrinsic signals such as second-harmonic generation by collagen- cells of interest must be fluorescently labeled by extrinsic probes or by expression of fluorescent proteins. The impression is thus created that these labeled cells are swimming in a black void; whereas in truth they are immersed in, and interact with a complex environment of structural elements and myriad other unlabeled, invisible cells.

In the six years since introduction of two-photon imaging to immunology, this technique has allowed researchers to address long-standing questions by directly visualizing in intact living tissues processes including cell-cell interactions, cell motility and localization, cellular signaling pathways and cytotoxic cell killing events. The field has rapidly evolved beyond initial phenomenological descriptions, and quantitative analyses together with computer modeling and simulation are now beginning to make sense of how the apparently chaotic cellular motions and interactions within lymph nodes lead to an efficient immune response. This progress is comprehensively reviewed in a recent publication1, which lists over 100 papers describing the application of two-photon microscopy for immunoimaging.

dissection and 2-photon imaging of peripheral lymph nodes in mice

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the initiation of an immune response 1. Here, we present methods for adoptive transfer of labeled T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node as first described in 2002 2. Two-photon imaging of immune cells requires that the cells are fluorescently labeled, either by staining with a cell tracker dye or by expressing a fluorescent protein. We demonstrate the adoptive transfer procedure of injecting cells derived from donor mice into the tail vein of a recipient animal, where they home to lymphoid organs within approximately 15-30 min. We illustrate the isolation of a lymph node and describe methods to ensure proper mounting of the excised lymph node. Other considerations such as proper oxygenation of perfused media, temperature, and laser power are discussed. Finally, we present 3D video images of naive CD4+ T cells exhibiting steady state motility at 37&deg;C.

Watch this Scientific Journal Video about Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice at JoVE.com

Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1. Here, we demonstrate adoptive transfer of T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph node.

Two-photon imaging has revealed an elegant choreography of motility and cellular interactions within the lymph node under basal conditions and at the ...

dissection-and-2-photon-imaging-of-peripheral-lymph-nodes-in-mice

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JoVE Journal

Biology

45.8K ビュー.  University of California, Irvine (UCI). 二光子イメージングには、基礎条件の下でリンパ節内および免疫応答1の間にリンパ球運動と細胞間相互作用を明らかにした。ここで、我々はT細胞、リンパ節の分離、およびCD4 +植リンパ節におけるT細胞のイメージング運動の養子移入を示しています。 

ビデオ: Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice

Two-photon imaging has uncovered lymphocyte motility and cellular interactions within the lymph node under basal conditions and durring an immune response 1.  Here, we demonstrate adoptive transfer of  T cells, isolation of lymph nodes, and imaging motility of CD4+ T cells in the explanted lymph...

isolation of cd4+ t cells from mouse lymph nodes using miltenyi macs purification

Isolation of CD4+ T cells from Mouse Lymph Nodes Using Miltenyi MACS Purification

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by...

intra-lymph node injection of biodegradable polymer particles

Intra-lymph Node Injection of Biodegradable Polymer Particles

Lymph nodes are the immunological tissues that orchestrate immune response and are a critical target for vaccines. Biomaterials have been employed to better target lymph nodes and to control delivery of antigens or adjuvants. This paper describes a technique combining these ideas to inject biocompatible polymer particles into lymph...

imaging of in situ interferon gamma production in the mouse spleen following listeria monocytogenes infection

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

Here, we describe a simple confocal imaging method to visualize the in situ localization of cells secreting the cytokine Interferon gamma in murine secondary lymphoid organs. This protocol can be extended for the visualization of other cytokines in diverse...

Imaging of In Situ Interferon Gamma Production in the Mouse Spleen following Listeria monocytogenes Infection

the bm12 inducible model of systemic lupus erythematosus (sle) in c57bl/6 mice

The bm12 Inducible Model of Systemic Lupus Erythematosus SLE in C57BL/6 Mice

The transfer of bm12 lymphocytes into a C57BL/6 recipient is an established model of systemic lupus erythematosus. Here we describe how to initiate disease using this model and how to characterize T follicular helper cells, germinal center B cells and plasma cells by flow...

The bm12 Inducible Model of Systemic Lupus Erythematosus (SLE) in C57BL/6 Mice

in vivo 2-photon calcium imaging in layer 2/3 of mice

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution...

high-frequency ultrasound imaging of mouse cervical lymph nodes

High-frequency Ultrasound Imaging of Mouse Cervical Lymph Nodes

This protocol describes the application of high-frequency ultrasound (HFUS) for imaging mouse cervical lymph nodes. This technique optimizes visualization and quantification of cervical lymph node morphology, volume and blood flow. Image-guided biopsy of cervical lymph nodes and processing of lymph tissue for histological evaluation is also...

a dissection method to harvest murine popliteal draining lymph nodes for ex vivo imaging

This video demonstrates a dissection technique to harvest popliteal draining lymph nodes from the mouse hind legs. Lymph nodes play a critical role in bridging innate and adaptive immunity, and harvesting intact lymph nodes &#8212; with a preserved tissue structure and cellular viability &#8212; can help study local and systemic immune...

A Dissection Method to Harvest Murine Popliteal Draining Lymph Nodes for Ex Vivo Imaging

Expose the popliteal fossa. The popliteal lymph node will appear as a translucent sphere within the fossa. Then, remove the lymph node with the microsurgery curved...

whole central and peripheral nervous system mice dissection

Whole Central and Peripheral Nervous System Mice Dissection

Here, we present a protocol to complete the dissection of central and peripheral nervous systems of mice. The dissected tissues are further analyzed in downstream applications, such as taking gross pictures or...

laparoscopic technique for serial collection of liver and mesenteric lymph nodes in macaques

Laparoscopic Technique for Serial Collection of Liver and Mesenteric Lymph Nodes in Macaques

Here, we describe a minimally invasive laparoscopic technique for serial sampling of liver and mesenteric lymph nodes (MLN) in macaques that allows for increased sampling frequency, and reduces the potential for surgical complications when compared to performing a...

non-surgical intratracheal instillation of mice with analysis of lungs and lung draining lymph nodes by flow cytometry

Non-surgical Intratracheal Instillation of Mice with Analysis of Lungs and Lung Draining Lymph Nodes by Flow Cytometry

We illustrate non-surgical delivery of test materials into the lungs of anesthetized mice via the trachea. This method permits lung exposure to bacterial and viral pathogens, cytokines, antibodies, beads, chemicals, or dyes. We further describe harvesting and processing of lungs and lung draining lymph nodes (LDLNs) for flow cytometry.

intravital imaging of the mouse thymus using 2-photon microscopy

Intravital Imaging of the Mouse Thymus using 2-Photon Microscopy

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...

generation of bone marrow derived murine dendritic cells for use in 2-photon imaging

Generation of Bone Marrow Derived Murine Dendritic Cells for Use in 2-photon Imaging

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

a cfse-based assay to monitor skin dendritic cell migration to draining lymph nodes in infected mice

This video demonstrates an assay for monitoring dendritic cell migration following footpad infection with Mycobacterium bovis Bacille Calmette-Gu&#233;rin BCG in mice. Upon bacterial infection, activated skin DC cells are labeled with a cell-permeable carboxyfluorescein diacetate succinimidyl ester (CFSE) dye. After a suitable interval for DC migration to the popliteal draining lymph node, the node cells are isolated and subjected to immunostaining for DC-specific surface markers.

A CFSE-Based Assay to Monitor Skin Dendritic Cell Migration to Draining Lymph Nodes in Infected Mice

in situ imaging of the mouse thymus using 2-photon microscopy

In situ Imaging of the Mouse Thymus Using 2-Photon Microscopy

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon...

micropuncture of bowman's space in mice facilitated by 2 photon microscopy

Micropuncture of Bowman&#039;s Space in Mice Facilitated by 2 Photon Microscopy

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

in vivo two-color 2-photon imaging of genetically-tagged reporter cells in the skin

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in...

collection and processing of lymph nodes from large animals for rna analysis: preparing for lymph node transcriptomic studies of large animal species

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

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...

leprdb mouse model of type 2 diabetes: pancreatic islet isolation and live-cell 2-photon imaging of intact islets

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several organisms including humans, non-human primates, rat and, as we describe here, mice. Magnetic bead-based cell separation allows for either positive selection (or cell depletion) as well as negative selection. Here, we demonstrate negative selection of untouched or na ve CD4+ helper T cells. Using this standard protocol we typically purify cells that are &#8805; 96% pure CD4+/CD3+. This protocol is used in conjunction with the protocol Dissection and 2-Photon Imaging of Peripheral Lymph Nodes in Mice published in issue 7 of JoVE, for purification of T cells and other cell types to adoptively transfer for imaging purposes. Although we did not demonstrate FACS analysis in this protocol video, it is highly recommended to check the overall purity of isolated cells using the appropriate antibodies via FACS. In addition, we demonstrate the non-sterile method of T cell isolation. If sterile cells are needed for your particular end-user application, be sure to do all of the demonstrated procedures in the tissue culture hood under standard sterile conditions. Thank you for watching and good luck with your own experiments!

Isolation of lymphocytes using the Miltenyi MACs kit is a reliable way to purify cells from whole lymphoid tissue homogenates. Cells purified using the Miltenyi system are typically &#8805; 96% pure. Here, we demonstrate the steps taken to isolate CD4+ T cells, one of the many kits offered by Miltenyi.

Isolation of cells from the primary source is a necessary step in many more complex protocols. Miltenyi offers kits to isolate cells from several ...

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development. To follow the migration of individuals in a crowd of thymocytes , we generate neonatal chimeras where less than one percent of the thymocytes are derived from a donor that is transgenic for a ubiquitously express fluorescent protein. To generate these partial hematopoetic chimeras, neonatal recipients are injected with bone marrow between 3-7 days of age. After 4-6 weeks, the mouse is sacrificed and the thymus is carefully dissected and bissected preserving the architecture of the tissue that will be imaged. The thymus is glued onto a coverslip in preparation for ex vivo imaging by TPM. During imaging the thymus is kept in DMEM without phenol red that is perfused with 95% oxygen and 5% carbon dioxide and warmed to 37&deg;C. Using this approach, we can study the events required for the generation of a diverse T cell repertoire.

We present step-by-step instructions for the generation of neonatal chimeras as well as the dissection and preparation of the thymus for ex vivo imaging by 2-Photon Microscopy.

Two-photon Microscopy (TPM) enables us to image deep into the thymus and document the events that are important for thymocyte development.  To follow the ...

A Method for 2-Photon Imaging of Blood Flow in the Neocortex through a Cranial Window

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is a powerful technique. Using in vivo 2-photon microscopy through a cranial window it is possible to image fluorescent dyes injected intravenously. This permits one to image the cortical vasculature and also to obtain measurements of blood flow. This technique was originally developed by David Kleinfeld and Winfried Denk. The method can be used to study blood flow dynamics during or after cerebral ischemia, in neurodegenerative disorders, in brain tumors, or in normal brain physiology. For example, it has been used to study how stroke causes shifts in blood flow direction and changes in red blood cell velocity or flux in and around the infarct. Here we demonstrate how to use 2-photon microscopy to image blood flow dynamics in the neocortex of living mice using fluorescent dyes injected into the tail vein.

Cortical blood flow dynamics can be studied in vivo by imaging fluorescent dextran dyes injected into the tail vein of rodents with 2-photon microscopy. This video shows how to image blood flow dynamics in neocortex of mice through a glass-covered cranial window preparation.

The ability to image the cerebral vasculature (from large vessels to capillaries) and record blood flow dynamics in the intact brain of living rodents is ...

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% CD11c high dendritic cells in culture that home to the draining lymph node after subcutaneous injection and present antigen to antigen specific T cells (see video). Additionally, we use Essen Instruments Incucyte to track dendritic cell maturation, where, at day 10, the morphology of the cultured cells is typical of a mature dendritic cell and &lt;85% of cells are CD11chigh. The study of antigen presentation in peripheral lymph nodes by 2-photon imaging revealed that there are three distinct phases of dendritic cell and T cell interaction1, 2. Phase I consists of brief serial contacts between highly motile antigen specific T cells and antigen carrying dendritic cells1, 2. Phase two is marked by prolonged contacts between antigen-specific T cell and antigen bearing dendritic cells1, 2. Finally, phase III is characterized by T cells detaching from dendritic cells, regaining motility and beginning to divide1, 2. This is one example of the type of antigen-specific interactions that can be analyzed by two-photon imaging of antigen-loaded cell tracker dye-labeled dendritic cells.

Antigen presentation in secondary lymphoid organs by dendritic cells is crucial for the initiation of the T cell mediated adaptive immune response. Here we demonstrate the culture of bone marrow derived murine dendritic cells, activation, and labeling for 2-photon imaging.

Several methods for the preparation of murine dendritic cells can be found in the literature. Here, we present a method that produces greater than 85% ...

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

Brown adipose tissue (BAT) differs from white adipose tissue (WAT) by its discrete location and a brown-red color due to rich vascularization and high density of mitochondria. BAT plays a major role in energy expenditure and non-shivering thermogenesis in newborn mammals as well as the adults 1. BAT-mediated thermogenesis is highly regulated by the sympathetic nervous system, predominantly via &beta; adrenergic receptor 2, 3. Recent studies have shown that BAT activities in human adults are negatively correlated with body mass index (BMI) and other diabetic parameters 4-6. BAT has thus been proposed as a potential target for anti-obesity/anti-diabetes therapy focusing on modulation of energy balance 6-8. While several cold challenge-based positron emission tomography (PET) methods are established for detecting human BAT 9-13, there is essentially no standardized protocol for imaging and quantification of BAT in small animal models such as mice. Here we describe a robust PET/CT imaging method for functional assessment of BAT in mice. Briefly, adult C57BL/6J mice were cold treated under fasting conditions for a duration of 4 hours before they received one dose of 18F-Fluorodeoxyglucose (FDG). The mice were remained in the cold for one additional hour post FDG injection, and then scanned with a small animal-dedicated micro-PET/CT system. The acquired PET images were co-registered with the CT images for anatomical references and analyzed for FDG uptake in the interscapular BAT area to present BAT activity. This standardized cold-treatment and imaging protocol has been validated through testing BAT activities during pharmacological interventions, for example, the suppressed BAT activation by the treatment of &beta;-adrenoceptor antagonist propranolol 14, 15, or the enhanced BAT activation by &beta;3 agonist BRL37344 16. The method described here can be applied to screen for drugs/compounds that modulate BAT activity, or to identify genes/pathways that are involved in BAT development and regulation in various preclinical and basic studies.

Functional Imaging of Brown Fat in Mice with 18F-FDG micro-PET/CT

A method of functional imaging of mouse brown adipose tissue (BAT) is described in which cold-stimulated uptake of 18F-Fluorodeoxyglucose (FDG) in BAT is non-invasively assessed with a standardized micro-PET/CT protocol. This method is robust and sensitive to detect differences in BAT activities in mouse models.

Brown adipose tissue (BAT) differs from white adipose  tissue (WAT) by its discrete location and a brown-red color due to rich  vascularization and high ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and diseases, such as obesity and fatty liver. Usually, lipids are stored in lipid droplets, which are the multifunctional lipid storage organelles in cells. Lipid droplets vary in size and number in different tissues and under different conditions. It has been reported that lipid droplets are tightly controlled through regulation of its biogenesis and degradation. In Drosophila melanogaster, the oenocyte is an important tissue for lipid metabolism and has been recently identified as a human liver analogue regarding lipid mobilization in response to stress. However, the mechanisms underlying the regulation of lipid droplet metabolism in oenocytes remain elusive. To solve this problem, it is of utmost importance to develop a reliable and sensitive method to directly visualize lipid droplet dynamic changes in oenocytes during development and under stressful conditions. Taking advantage of the lipophilic BODIPY 493/503, a lipid droplet-specific fluorescent dye, described here is a detailed protocol for the dissection and subsequent lipid droplet staining in the oenocytes of Drosophila larvae in response to starvation. This allows for qualitative analysis of lipid droplet dynamics under various conditions by confocal microscopy. Furthermore, this rapid and highly reproducible method can also be used in genetic screens for indentifying novel genetic factors involving lipid droplet metabolism in oenocytes and other tissues.

Dissection and Lipid Droplet Staining of Oenocytes in Drosophila Larvae

Presented here are detailed methods for the dissection and lipid droplet staining of oenocytes in Drosophila larvae using BODIPY 493/503, a lipid droplet-specific fluorescent dye.

Lipids are essential for animal development and physiological homeostasis. Dysregulation of lipid metabolism results in various developmental defects and ...