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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N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunological+properties+of+an+antibody+containing+a+fluorescent+group.">Immunological properties of an antibody containing a fluorescent group.</a> Proceedings of the Society for Experimental Biology and Medicine. 47 (2), 200-202 (1941).</li><li>Lerario, A. M., Finco, I., LaPensee, C., Hammer, G. 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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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	<tbody>
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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>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Disposable needles 25Gx5/8&#34;</td>
			<td style="width:156px;">Exel International</td>
			<td style="width:119px;">26403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Paraformaldehyde (PFA)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P6148</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylene</td>
			<td style="width:156px;">Fisher Chemical</td>
			<td style="width:119px;">X5P1GAL&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">200 Proof Ethanol</td>
			<td style="width:156px;">Decon Labs, Inc.</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
			<td style="width:119px;"></td>
			<td style="width:167px;">Light source</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Coolsnap Myo</td>
			<td style="width:156px;">Photometrics</td>
			<td style="width:119px;"></td>
			<td>Camera</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Optiphot-2&#160;</td>
			<td style="width:156px;">Nikon</td>
			<td style="width:119px;"></td>
			<td>Microscope</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">microtome</td>
			<td style="width:156px;">Americal Optical</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tissue processor&#160;</td>
			<td style="width:156px;">Leica</td>
			<td style="width:119px;">ASP300S</td>
			<td></td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="42">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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>
		</tr>
	</tbody>
</table>

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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23.0K Views.  University of Michigan Health System. 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 gl...