Name: isolation, fixation, and immunofluorescence imaging of mouse adrenal glands
Uploaded: 2018-10-02T14:45:00
Description: Watch this Scientific Journal Video about Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands at JoVE.com

We thank Dr. Mohamad Zubair for his helpful suggestions and technical assistance in the establishment of this protocol. This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health Research Grant 2R01-DK062027 (to G.D.H).

All methods were performed in accordance with institutionally approved protocols under the auspice of the University Committee on Use and Care of Animals at the University of Michigan.
1. Preparation for Surgery
<ol>
	<li>On the day prior to surgery, prepare 4% paraformaldehyde (PFA)/phosphate buffered saline (PBS). In case of frozen aliquots, proceed to thaw one and store at 4 &#176;C. 
	NOTE: 4% PFA is not stable for more than 48 h. 
	CAUTION: PFA is toxic, avoid contact with ski...

<ol>
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This protocol describes a method for the isolation of mouse adrenal glands together with the preparation and staining of sectioned paraffin-embedded mouse adrenals.
Compared to other protocols we tested, this immunofluorescence protocol has proven suitable for the majority of antibodies used in our laboratory. However, in certain cases it may require some adjustments to improve the staining results. One variable that can easily be modified and tested is the length of fixation. In our laborator...

Immunohistochemistry is a technique for detecting tissue components with the use of antibodies to specific cellular molecules and subsequent staining techniques to detect the conjugated antibodies1. This immunohistochemical procedure requires specific fixation and processing of tissues that are often empirically determined for the specific antigen, tissue and antibody utilized2. Fixation is crucial to preserve the &#34;original&#34; state of the tissue and thereby maintaining intact cellular and subcellular structures and expression patterns. Further processing and embedding procedures are required to prepare the tissue for sectioning into thin slices that are used for histologic studies involving immunohistochemistry.
Immunostaining can be performed with either chromogenic or fluorescent detection. Chromogenic detection requires the utilization of an enzyme to convert a soluble substrate into an insoluble colored product. While this enzyme can be conjugated to the antibody recognizing the antigen (primary antibody), it is more often conjugated to the antibody recognizing the primary antibody (i.e., the secondary antibody). This technique is highly sensitive; the colored product resulting from the enzymatic reaction is photostable and requires only a brightfield microscope for imaging. However, chromogenic immunostaining may not be suitable when trying to visualize two proteins that co-localize, since the deposition of one color can mask the deposition of the other one. In the case of co-staining, immunofluorescence has proven to be more advantageous. The advent of immunofluorescence is attributed to Albert Coons and colleagues, who developed a system to identify tissue antigens with antibodies marked with fluorescein and visualize them in the sectioned tissues under ultraviolet light3. Fluorescence detection is based on an antibody conjugated with a fluorophore that emits light after excitation. Because there are several fluorophores with emissions at different wavelengths (with no or little overlap), this detection method is ideal for the studies of multiple proteins.
The adrenal gland is a paired organ located above the kidney and characterized by two embryologically distinct components surrounded by a mesenchymal capsule. The outer adrenal cortex, derived from the mesonephric intermediate mesoderm, secretes steroid hormones while the inner medulla, derived from the neural crest, produces catecholamines including adrenaline, noradrenaline, and dopamine. The adrenal cortex is histologically and functionally divided in three concentric zones, with each zone secreting different classes of steroid hormones: the outer zona glomerulosa (zG) produces mineralocorticoids that regulate electrolyte homeostasis and intravascular volume; the middle zona fasciculata (zF), directly beneath the zG, secretes glucocorticoids that mediate the stress response through the mobilization of energy stores to increase plasma glucose; and the inner zona reticularis (zR), which synthesizes sex steroid precursors (i.e., dehydroepiandrosterone (DHEAS))4.
Some variation in adrenocortical zonation is present between species: for example, Mus musculus lacks the zR. The unique postnatal X-zone of M. musculus is a remnant of the fetal cortex characterized by small lipid-poor cells with acidophilic cytoplasms5. The X-zone disappears at puberty in male mice and after the first pregnancy in female mice, or gradually degenerates in not-bred females6,7. Moreover, the tortuosity and thickness of the zG exhibits marked variation between species as does organization of peripheral stem and progenitor cells in and adjacent to the zG. The rat, unlike other rodents, has a visible undifferentiated zone (zU) between the zG and zF that functions as a stem cell zone and/or a zone of transient amplifying progenitors. Whether the zU is unique to rats or simply a more prominently organized cluster of cells is unknown8,9.
Cells of the adrenal cortex contain lipid droplets that store cholesterol esters that serve as the precursor of all steroid hormones10,11.&#160; The term &#34;steroidogenesis&#34; defines the process of production of steroid hormones from cholesterol via a series of enzymatic reactions that involve the activity of steroidogenic factor 1 (SF1), whose expression is a marker of steroidogenic potential. In the adrenal gland, Sf1 expression is present only in cells of the cortex12. An interesting study found the expression of endogenous biotin in adrenocortical cells with steroidogenic potential13. While this can be the cause of a higher background in biotin/streptavidin-based staining methods, due to the detection of endogenous biotin by antibody conjugated with streptavidin, this characteristic could be also employed to distinguish the steroidogenic cells from other populations within the adrenal gland, i.e., endothelial, capsular, and medulla cells.
Innervated by sympathetic preganglionic neurons, the adrenal medulla is characterized by basophilic cells with a granular cytoplasm containing epinephrine and norepinephrine. Medulla cells are named &#34;chromaffin&#34; due to the high content of catecholamines that form a brown pigment after oxidation14. Tyrosine hydroxylase (TH) is the enzyme that catalyzes the rate-limiting step in the synthesis of catecholamines and, in the adrenal gland, is expressed only in the medulla15.
Here we present a protocol for the isolation of mouse adrenal glands, their processing for embedding in paraffin and sectioning, and a method to perform immunofluorescence staining on adrenal sections in order to identify the cellular types constituting the adrenal cortex and medulla. This protocol is a standard in our laboratory for immunostaining with multiple antibodies routinely used in our research.

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			<td height="42" style="height:42px;width:216px;">24-well cell culture plate</td>
			<td style="width:156px;">Nest Biotechnology Co.</td>
			<td style="width:119px;">0412B</td>
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			<td height="21" style="height:21px;width:216px;">Disposable needles 25Gx5/8&#34;</td>
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			<td style="width:119px;">26403</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P6148</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Paraplast plus&#160;</td>
			<td style="width:156px;">McCormik scientific</td>
			<td style="width:119px;">39502004</td>
			<td>Paraffin for tissue embedding</td>
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			<td height="42" style="height:42px;width:216px;">Shandon biopsy cassettes II with attached lid</td>
			<td style="width:156px;">Thermo scientific</td>
			<td style="width:119px;">1001097</td>
			<td>Cassettes for tissue processing</td>
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			<td height="21" style="height:21px;width:216px;">High Profile Microtome Blades</td>
			<td style="width:156px;">Accu-Edge</td>
			<td style="width:119px;">4685</td>
			<td>Disposable stainless steel blades</td>
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			<td height="42" style="height:42px;width:216px;">Peel-a-way disposable plastic tissue embedding molds</td>
			<td style="width:156px;">Polysciences Inc.</td>
			<td style="width:119px;">18986</td>
			<td>Truncated,22mm square top tapered to 12mm bottom</td>
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			<td height="42" style="height:42px;width:216px;">Superfrost Plus Microscope Slides</td>
			<td style="width:156px;">Fisherbrand</td>
			<td style="width:119px;">12-550-15</td>
			<td>75x25x1 mm</td>
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			<td height="21" style="height:21px;width:216px;">Xylene</td>
			<td style="width:156px;">Fisher Chemical</td>
			<td style="width:119px;">X5P1GAL&#160;</td>
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			<td height="21" style="height:21px;width:216px;">200 Proof Ethanol</td>
			<td style="width:156px;">Decon Labs, Inc.</td>
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			<td height="42" style="height:42px;width:216px;">Certi-Pad Gauze pads&#160;</td>
			<td style="width:156px;">Certified Safety Mfg, Inc</td>
			<td style="width:119px;">231-210</td>
			<td>3&#34;x3. Sterile latex free gauze pads</td>
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			<td height="21" style="height:21px;width:216px;">M.O.M kit&#160;</td>
			<td style="width:156px;">Vector laboratories</td>
			<td style="width:119px;">BMK-2202</td>
			<td>For detecting mouse primary antibodies on mouse tissue</td>
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			<td height="21" style="height:21px;width:216px;">KimWipes</td>
			<td style="width:156px;">Kimtech</td>
			<td style="width:119px;">34155</td>
			<td>Wipes 4.4x8.4 inch</td>
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			<td height="21" style="height:21px;width:216px;">Super PAP PEN</td>
			<td style="width:156px;">Invitrogen&#160;</td>
			<td style="width:119px;">00-8899</td>
			<td>Pen to draw on slides</td>
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			<td height="21" style="height:21px;width:216px;">Microscope cover glass&#160;</td>
			<td style="width:156px;">Fisherbrand</td>
			<td style="width:119px;">12-544-D</td>
			<td>Size: 22x50x1.5</td>
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			<td height="21" style="height:21px;width:216px;">DAPI</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">D9542&#160;</td>
			<td>(Prepared in 20mg/mL stock)</td>
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			<td height="21" style="height:21px;width:216px;">ProLong Gold antifade reagent</td>
			<td style="width:156px;">Molecular Probes</td>
			<td style="width:119px;">P36930</td>
			<td>Mounting agent for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">X-cite series 120Q</td>
			<td style="width:156px;">Lumen Dynamics</td>
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			<td style="width:167px;">Light source</td>
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			<td height="21" style="height:21px;width:216px;">Coolsnap Myo</td>
			<td style="width:156px;">Photometrics</td>
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			<td>Camera</td>
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			<td height="21" style="height:21px;width:216px;">Optiphot-2&#160;</td>
			<td style="width:156px;">Nikon</td>
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			<td>Microscope</td>
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			<td height="21" style="height:21px;width:216px;">microtome</td>
			<td style="width:156px;">Americal Optical</td>
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			<td height="21" style="height:21px;width:216px;">Tissue embedder</td>
			<td style="width:156px;">Leica</td>
			<td style="width:119px;">EG1150 H&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Tissue processor&#160;</td>
			<td style="width:156px;">Leica</td>
			<td style="width:119px;">ASP300S</td>
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			<td height="21" style="height:21px;width:216px;">Normal goat serum</td>
			<td style="width:156px;">Sigma</td>
			<td style="width:119px;">G9023</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">Mouse anti-TH</td>
			<td style="width:156px;">Millipore</td>
			<td style="width:119px;">MAB318</td>
			<td>Primary antibody</td>
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			<td height="21" style="height:21px;width:216px;">Rabbit anti-SF1</td>
			<td>Ab proteintech group (PTGlabs)</td>
			<td style="width:119px;">&#160;custom made</td>
			<td>Primary antibody</td>
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			<td height="42" style="height:42px;width:216px;">Alexa-488 Mouse IgG&#160; raised goat</td>
			<td style="width:156px;">Jackson ImmunoResearch</td>
			<td>115-545-003&#160;</td>
			<td>Secondary antibody</td>
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			<td height="42" style="height:42px;width:216px;">Dylight-549 Rabbit IgG raised goat</td>
			<td style="width:156px;">Jackson ImmunoResearch</td>
			<td>111-505-003</td>
			<td>Secondary antibody</td>
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			<td height="21" style="height:21px;width:216px;">Citrate acid anhydrous</td>
			<td style="width:156px;">Fisher Chemical</td>
			<td style="width:119px;">A940-500</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">NIS-Elements Basic Research&#160;</td>
			<td style="width:156px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Software for imaging</td>
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isolation, fixation, and immunofluorescence imaging of mouse adrenal glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Figure 1&#160;represents a schematic of the entire protocol described above. Adrenal glands are harvested from mice, adjacent adipose tissue is removed under a dissecting microscope, and the adrenal are then fixed in 4% PFA. After this step, adrenals are processed and embedded in paraffin, and sectioned with a microtome to cut the organ into thin slices that are deposited on microscope slides. After drying of the sections, immunofluorescence is carried out an...

Watch this Scientific Journal Video about Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands at JoVE.com

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

Immunofluorescence is a basic method that can help answer key questions in the adrenal field about adrenal zonation, cell identity and function. The main advantage of this technique is that it allows the study of multiple proteins while preserving the morphology of the adrenal tissue. To remove the murine adrenal gland, cut around the surrounding adipose tissue and place the gland in one well of a 24-multiwell plate containing fresh PBS on ice.Transfer the gland onto a Petri dish or glass support under a dissecting microscope and use two 25 gauge needles to remove the adjacent fat tissue. When all of the fat has been removed, fix the tissue in 4%paraformaldehyde at four degrees Celsius with rocking for two hours. At the end of the incubation, rinse the gland with three 15-minute washes at four degrees Celsius in one to two milliliters of fresh PBS per wash.After the last wash, dehydrate the gland in two sequential ethanol immersions at four degrees Celsius with rocking for two hours per incubation. To prepare the adrenal tissue for processing, wrap the gland in gauze and enclose the adrenal in a tissue-embedding cassette. Then, place the cassette in a jar filled with 70%ethanol for four degrees Celsius storage until processing.To process the tissue, insert the cassette into the tissue processor for processing as indicated in the table. At the end of the program, transfer the cassette into an embedding station filled with melted 65 degree paraffin and use a pair of forceps to unwrap the gauze. Gently transfer the adrenal in the appropriate position at the center of the base of the embedding mold and pour the melted paraffin onto the gland.Then, carefully move the embedding mold to a cooling tray to facilitate hardening of the paraffin. Before sectioning by rotary microtome, label a series of microscope slides and lay the slides on a 37 degree Celsius slide warmer. Dispense about 450 microliters of autoclaved type one ultra pure water onto each slide and set the section cutting thickness of the microtome to five micrometers.Load the embedded tissue onto the cutting block and lower the paraffin block until the sample is at face level with the edge of the microtome blade. Rotating the hand wheel with a steady rhythm, perform an initial trimming to remove the paraffin and expose the adrenal tissue. Then, acquire five micrometer sections through the end of the adrenal gland using forceps to transfer each section into the water droplet onto one of the pre-labeled slides as it is collected.After confirming the presence of tissue in the sections by microscopy, dry the slides on the hot plate for about two hours and bake them at least overnight on a slide tray at 37 degrees Celsius until no more water is visible. Once dry, store the slides in a slide box at room temperature. For immunofluorescence antigen retrieval, de-paraffinize the slides with two five-minute 100%xylene immersions, followed by rehydration in a descending ethanol and water immersion series.After the water immersion, transfer the slides to a metal slide holder and place the holder in a beaker of boiling 0.01 molar citrate on a hot plate for 10 minutes. At the end of the incubation, remove the beaker from the hot plate and let it cool for 20 minutes, taking care that all of the adrenal sections are still covered with buffer. When the beaker is cooled, transfer the slides into a Coplin jar for three 10-minute washes in fresh PBS with gentle rocking.After the last wash, carefully wipe the surface of each slide without damaging the tissue sections and use a hydrophobic marker to make a circle around each section. Place the slides in a humidified chamber and quickly add at least 50 microliters of blocking solution to each section for a one-hour incubation at room temperature. At the end of the blocking incubation, wash the slides three times in fresh PBS for five minutes per wash with rocking and return slides to the chamber.Next, add the primary antibody cocktail of interest to each section for an overnight incubation at four degrees Celsius. The next morning, wash the samples with three 15-minute washes in fresh PBS per wash and label the slides with the appropriate secondary antibody solution for one hour protected from light. At the end of the incubation, wash the samples with three 15-minute washes on a rocker protected from light, labeling the nuclei of the sections with DAPI for seven minutes at room temperature protected from light after the last wash.After three five-minute PBS washes with rocking, add 60 microliters of mounting agent suitable for immunofluorescence along the surface of one cover glass per sample and lightly press each slide tissue section side down onto a cover glass, taking care to avoid bubbles. Then, lay each slide flat in a dark container for curing at room temperature for at least 24 hours before imaging. During the fat removal process, it is critical not to let the adrenal gland dry out as dehydration can damage the tissue structure.Following a successful removal of the surrounding adipose tissue, the adrenal gland should be easily detectable with no extra visible fat. Immunostaining of adrenal gland sections as demonstrated reveals nuclear steroidogenic factor one staining of the adrenocortical cells, tyrosine hydroxylase staining of the medullary cells, DAPI staining of the nuclei of non-steroidogenic cells of the outer capsule, and co-staining with adrenocortical and medullary cells. When attempting this procedure, it is important to remember that mouse adrenals are small and that their positions are somewhat variable making their detection difficult.Therefore, it is crucial to have a clear view of the area during the dissection. These procedures are basic techniques for the study of the adrenal gland in vivo. Following these, additional methods of immunostaining can be performed to meet specific scientific needs.After its development, immunostaining paved the way for researchers in the biomedical science fields to explore in situ protein expression, a useful tool in many disciplines and fields.

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JoVE Core

JoVE Journal

Anatomy and Physiology

Immunology and Infection

Unit 1

The Endocrine System

SCIENTISTS IN ACTION

Isolation of Mouse Peritoneal Cavity Cells

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal tract and other viscera. It harbors a number of immune cells including macrophages, B cells and T cells. The presence of a high number of na&iuml;ve macrophages in the peritoneal cavity makes it a preferred site for the collection of na&iuml;ve tissue resident macrophages (1). The peritoneal cavity is also important to the study of B cells because of the presence of a unique peritoneal cavity-resident B cell subset known as B1 cells in addition to conventional B2 cells. B1 cells are subdivided into B1a and B1b cells, which can be distinguished by the surface expression of CD11b and CD5. B1 cells are an important source of natural IgM providing early protection from a variety of pathogens (2-4). These cells are autoreactive in nature (5), but how they are controlled to prevent autoimmunity is still not understood completely. On the contrary, CD5+ B1a cells possess some regulatory properties by virtue of their IL-10 producing capacity (6). Therefore, peritoneal cavity B1 cells are an interesting cell population to study because of their diverse function and many unaddressed questions associated with their development and regulation. The isolation of peritoneal cavity resident immune cells is tricky because of the lack of a defined structure inside the peritoneal cavity. Our protocol will describe a procedure for obtaining viable immune cells from the peritoneal cavity of mice, which then can be used for phenotypic analysis by flow cytometry and for different biochemical and immunological assays.

The peritoneal cavity in mammals contains different immune cell populations crucial for innate immune responses. An efficient isolation method is required for biochemical and functional analyses of these cells. Here we provide a comprehensive method for the isolation of peritoneal cavity cells in the mouse.

The peritoneal cavity is a membrane-bound and fluid-filled abdominal cavity of mammals, which contains the liver, spleen, most of the gastro-intestinal ...

Isolation of Mouse Lung Dendritic Cells

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the respiratory tract. At least three main subsets of lung dendritic cells have been described in mice: conventional DC (cDC) 4, plasmacytoid DC (pDC) 5 and the IFN-producing killer DC (IKDC) 6,7. The cDC subset is the most prominent DC subset in the lung 8. 
The common marker known to identify DC subsets is CD11c, a type I transmembrane integrin (&beta;2) that is also expressed on monocytes, macrophages, neutrophils and some B cells 9. In some tissues, using CD11c as a marker to identify mouse DC is valid, as in spleen, where most CD11c+ cells represent the cDC subset which expresses high levels of the major histocompatibility complex class II (MHC-II). However, the lung is a more heterogeneous tissue where beside DC subsets, there is a high percentage of a distinct cell population that expresses high levels of CD11c bout low levels of MHC-II. Based on its characterization and mostly on its expression of F4&#47;80, an splenic macrophage marker, the CD11chiMHC-IIlo lung cell population has been identified as pulmonary macrophages 10 and more recently, as a potential DC precursor 11. 
In contrast to mouse pDC, the study of the specific role of cDC in the pulmonary immune response has been limited due to the lack of a specific marker that could help in the isolation of these cells. Therefore, in this work, we describe a procedure to isolate highly purified mouse lung cDC. The isolation of pulmonary DC subsets represents a very useful tool to gain insights into the function of these cells in response to respiratory pathogens as well as environmental factors that can trigger the host immune response in the lung.

A highly purified preparation of mouse lung dendritic cells is described. Specific emphasis is given to the isolation of conventional dendritic cell subset.

Lung dendritic cells (DC) play a fundamental role in sensing invading pathogens 1,2 as well as in the control of tolerogenic responses 3 in the ...

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

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

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

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

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

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.

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

Isolation and Characterization of Dendritic Cells and Macrophages from the Mouse Intestine

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and food antigens while concomitantly remaining poised to mount inflammatory responses toward invasive pathogens1,2. Antigen presenting cells, particularly DCs and macrophages, play critical roles in maintaining intestinal immune homeostasis via their ability to sense and appropriately respond to the microbiota3-14. Efficient isolation of intestinal DCs and macrophages is a critical step in characterizing the phenotype and function of these cells. While many effective methods of isolating intestinal immune cells, including DCs and macrophages, have been described6,10,15-24, many rely upon long digestions times that may negatively influence cell surface antigen expression, cell viability, and/or cell yield. Here, we detail a methodology for the rapid isolation of large numbers of viable, intestinal DCs and macrophages. Phenotypic characterization of intestinal DCs and macrophages is carried out by directly staining isolated intestinal cells with specific fluorescence-labeled monoclonal antibodies for multi-color flow cytometric analysis. Furthermore, highly pure DC and macrophage populations are isolated for functional studies utilizing CD11c and CD11b magnetic-activated cell sorting beads followed by cell sorting.

Here, we detail a methodology for the rapid isolation of mouse intestinal dendritic cells (DCs) and macrophages. Phenotypic characterization of intestinal DCs and macrophages is performed using multi-color flow cytometric analysis while magnetic bead enrichment followed by cell sorting is used to yield highly pure populations for functional studies.

Within the intestine reside unique populations of innate and adaptive immune cells that are involved in promoting tolerance towards commensal flora and ...

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

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

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

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

Isolation of Lymphocytes from Mouse Genital Tract Mucosa

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and bacteria 1. Mucosae are also inductive sites in the host to generate immunity against pathogens, such as the Peyers patches in the intestinal tract and the nasal-associated lymphoreticular tissue in the respiratory tract. This unique feature brings mucosal immunity as a crucial player of the host defense system. Many studies have been focused on gastrointestinal and respiratory mucosal sites. However, there has been little investigation of reproductive mucosal sites. The genital tract mucosa is the primary infection site for sexually transmitted diseases (STD), including bacterial and viral infections. STDs are one of the most critical health challenges facing the world today. Centers for Disease Control and Prevention estimates that there are 19 million new infectious every year in the United States. STDs cost the U.S. health care system $17 billion every year 2, and cost individuals even more in immediate and life-long health consequences. In order to confront this challenge, a greater understanding of reproductive mucosal immunity is needed and isolating lymphocytes is an essential component of these studies. Here, we present a method to reproducibly isolate lymphocytes from murine female genital tracts for immunological studies that can be modified for adaption to other species. The method described below is based on one mouse.&#x2003;

An efficient way to isolate lymphocytes from mouse genital tract is described. This method takes advantage of enzyme digestion and Percoll gradient separation to allow efficient isolation. This technique is also adaptable to for use in other species

Mucosal surfaces, including in the gastrointestinal, urogenital, and respiratory tracts, provide portals of entry for pathogens, such as viruses and ...

Time-lapse 3D Imaging of Phagocytosis by Mouse Macrophages

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of the actin cytoskeleton to capture, envelop and engulf large particles. Although phagocytic receptors, downstream signaling pathways, and effectors, such as Rho GTPases, have been identified, the dynamic cytoskeletal remodeling of specific receptor-mediated phagocytic events remain unclear. Four decades ago, two distinct mechanisms of phagocytosis, exemplified by Fc&#947; receptor (Fc&#947;R)- and complement receptor (CR)-mediated phagocytosis, were identified using scanning electron microscopy. Binding of immunoglobulin G (IgG)-opsonized particles to Fc&#947;Rs triggers the protrusion of thin membrane extensions, which initially form a so-called phagocytic cup around the particle before it becomes completely enclosed and retracted into the cell. In contrast, complement opsonized particles appear to sink into the phagocyte following binding to complement receptors. These two modes of phagocytosis, phagocytic cup formation and sinking in, have become well established in the literature. However, the distinctions between the two modes have become blurred by reports that complement receptor-mediated phagocytosis may induce various membrane protrusions. With the availability of high resolution imaging techniques, phagocytosis assays are required that allow real-time 3D (three dimensional) visualization of how specific phagocytic receptors mediate the uptake of individual particles. More commonly used approaches for the study of phagocytosis, such as end-point assays, miss the opportunity to understand what is happening at the interface of particles and phagocytes. Here we describe phagocytic assays, using time-lapse spinning disk confocal microscopy, that allow 3D imaging of single phagocytic events. In addition, we describe assays to unambiguously image Fc&#947; receptor- or complement receptor-mediated phagocytosis.

Here we describe methods using spinning disk confocal microscopy to image single phagocytic events by mouse resident peritoneal macrophages. The protocols can be extended to other phagocytic cells.

Phagocytosis plays a key role in host defense, as well as in tissue development and maintenance, and involves rapid, receptor-mediated rearrangements of ...

Isolation and Quantitative Evaluation of Brush Cells from Mouse Tracheas

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. They are part of a family of chemosensory epithelial cells which include tuft cells in the intestinal mucosa, brush cells in the trachea, and solitary chemosensory and microvillous cells in the nasal mucosa. Chemosensory cells in different epithelial compartments share key intracellular markers and a core transcriptional signature, but also display significant transcriptional heterogeneity, likely reflective of the local tissue environment. Isolation of tracheal brush cells from single cell suspensions is required to define the function of these rare epithelial cells in detail, but their isolation is challenging, potentially due to the close interaction between tracheal brush cells and nerve endings or due to airway-specific composition of tight and adherens junctions. Here, we describe a procedure for isolation of brush cells from mouse tracheal epithelium. The method is based on an initial separation of tracheal epithelium from the submucosa, allowing for a subsequent shorter incubation of the epithelial sheet with papain. This procedure offers a rapid and convenient solution for flow cytometric sorting and functional analysis of viable tracheal brush cells.

Brush cells are rare cholinergic chemosensory epithelial cells found in the na&#239;ve mouse trachea. Due to their limited numbers, ex vivo evaluation of their functional role in airway immunity and remodeling is challenging. We describe a method for isolation of tracheal brush cells by flow cytometry.

Tracheal brush cells are cholinergic chemosensory epithelial cells poised to transmit signals from the airway lumen to the immune and nervous systems. ...

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

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

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

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