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 skin and eyes, and dispose in an appropriate container.</li>
	<li>On the day of the surgery, prepare the surgical instruments by sterilizing the scissors and forceps with 70% ethanol (EtOH) and let them dry on a paper towel.</li>
	<li>Place a 24-well plate filled with 1x PBS (1 mL/well is sufficient) in an ice bucket. 
	NOTE: Adrenals will be kept in this multi-well culture dish after surgery.</li>
</ol>
2. Adrenal Dissection
<ol>
	<li>Euthanize the mouse following standard protocols approved by the Institutional Animal Care and Use Committee (IACUC). A secondary method of euthanasia to ensure death (for example decapitation) is required.
	<ol>
		<li>For euthanasia by isoflurane overdose, place the mouse in a chamber filled with isoflurane vapors until respiration ceases, then remove the mouse from the chamber, lay it on a surface and perform cervical dislocation. 
		NOTE: Most euthanasia methods cause distress to the animal and are not suitable for stress studies because they may add the confounding variable of activation of the pituitary-adrenal axis and medullary catecholamine release. In this instance, decapitation is the suggested technique to adopt.</li>
	</ol>
	</li>
	<li>Lay the mouse supine, sterilize the incision area on the abdomen and flanks with 70% EtOH. 
	NOTE: While shaving the fur will increase sterility, for this particular application it is not necessary.</li>
	<li>Cut the skin in the middle of the abdomen using scissors, separate the skin from the peritoneum, deskin the mouse by pinching the skin around the cut and pulling it in two opposite directions (caudal and rostral). Then cut the peritoneum until reaching the posterior left and right flanks.</li>
	<li>Remove the murine adrenal gland cutting around the surrounding adipose tissue and place it in the 24-multiwell plate filled with 1x PBS and kept on ice.</li>
</ol>
3. Peri-adrenal Fat Removal
<ol>
	<li>Under a dissecting microscope, place the adrenal on a glass base or a Petri-dish on the stage plate, position the light source, select the magnification and adjust the focus to obtain a clear view of the entire adrenal. 
	NOTE: The magnification used can vary depending on personal preferences. A total magnification of 30X with a plan lens 1X, zoom 3X, and eyepiece 10X allows to visualize&#160;the whole gland with a good field of view.</li>
	<li>Quickly remove the adjacent fat tissues with two 25 G needles, avoiding dehydration of the adrenal. If this happens before all fat is removed, move the adrenal back into the well containing 1x PBS for 1&#8211;2 min, and then continue with the fat removal process.</li>
	<li>Wash 2x for 2 min each in 1x PBS.</li>
</ol>
4. Tissue Processing and Embedding
<ol>
	<li>Fix the tissues in 4% PFA/PBS at 4 &#176;C on a rocking platform for 2 h.</li>
	<li>Remove the 4% PFA and wash the tissues with 1x PBS, 3x for 15 min each at 4 &#176;C. 
	CAUTION: PFA is toxic and is a hazardous waste; it should be handled with caution under a chemical hood and disposed into a hazardous waste container.</li>
	<li>Incubate the adrenals in 50% EtOH at 4 &#176;C on a rocking platform for 2 h.</li>
	<li>Incubate the adrenals in 70% EtOH at 4 &#176;C on a rocking platform for a minimum of 2 h. 
	NOTE: If not proceeding with tissue processing for embedding, adrenals can be stored in 70% EtOH at 4 &#176;C. Avoiding long-term storage in 70% EtOH and proceeding to tissue processing as soon as possible yields better quality samples.</li>
	<li>Prepare the adrenal for tissue processing by wrapping it in gauze and enclosing it in a tissue embedding cassette. Place the cassette in a jar filled with 70% EtOH and store at 4 &#176;C until ready to move to the tissue processor. Insert the cassette into the tissue processor programmed as reported in Table 1 and run the program.</li>
	<li>Transfer the cassette into an embedding station filled with melted paraffin at 65 &#176;C. If a cooling station is not available, position a cold cooling tray next to it.</li>
	<li>Label an embedding mold and open the tissue embedding cassette. Using a pair of forceps, unwrap the gauze and place the adrenal gently in the desired position at the center of the base in the embedding mold. Pour the melted paraffin on the adrenal. If necessary, hold the adrenal with forceps to secure its position in the mold otherwise it may move around inside the mold.</li>
	<li>Gently move the embedding mold to the cooling tray and wait until the hardening of the paraffin. After that, to facilitate sectioning, store embedding molds in a cool place.</li>
</ol>
5. Sectioning with a Rotary Microtome
<ol>
	<li>Check that the microtome is clean before starting, brush away all discarded paraffin residues, ensure that the knife (or blade) is sharpened, oil the internal mechanism and fully retract the block holder if necessary.</li>
	<li>Label a series of microscope slides (positively charged or coated to prevent tissue loss (see Table of Materials)), lay them on a slide warmer set at 37 &#176;C, and dispense some autoclaved type 1 ultrapure water on top of each slide.</li>
	<li>Set section thickness at 5 &#181;m.</li>
	<li>Insert the microtome blade in the blade holder, control the angle of the blade, make sure that the blade is firmly secured into the holder and check the clearance of the paraffin block and block holder.</li>
	<li>Bring the block holder to its highest position and, if possible, lock the operating handle, remove the paraffin block from the mold and place it into the block holder securing it firmly with the clamp. The angle of the center line of the blade with the facing surface of the block should be about 20&#176;. If necessary, readjust the paraffin block. 
	CAUTION: The blade is sharp.</li>
	<li>If previously locked, unlock the hand wheel and lower the paraffin block until its face is level with the edge of the microtome blade. Rotate the hand wheel with a steady rhythm. Perform initial trimming to remove the paraffin and to expose the adrenal.</li>
	<li>Keep turning the hand wheel, and section until&#160;the end of the adrenal. Use a brightfield or dissecting microscope to check for the presence of tissue in the sections. Detach the ribbon from the blade and with the help of another pair of forceps place it on the water laid on the glass slide. It may be useful to place the ribbon on a surface, stretch it carefully, and then mount it on the slide.</li>
	<li>Let the slides dry on the hot plate. Then lay them flat on a slide tray and bake at 37 &#176;C overnight or longer if water is still present. Once dry, store the slides in a slide box at room temperature.</li>
	<li>Put away all the used equipment and clean the microtome.</li>
</ol>
6. Immunofluorescence
<ol>
	<li>Select the slides for immunostaining and place them in a slide holder.</li>
	<li>On a hot plate, bring a beaker filled with 0.01M citrate at pH 2.0 to boil. The volume of the buffer depends on the beaker size and must be enough for covering the sections on the slides during boiling (some evaporation needs to be taken into account). Cover the beaker with aluminum foil. 
	NOTE: Other methods for antigen retrieval are available (see Discussion).</li>
	<li>Deparaffinize the slides in 100% xylene 2x for 5 min each. Rehydrate the slides using the following series: 100% EtOH 2x for 5 min, 70% EtOH 2x for 5 min, type 1 ultrapure water for 5 min. 
	NOTE: After deparaffinization, it is important to not let the sections dry out: tissue sections must always be covered with buffers or solutions until the end of the procedure or the tissue structure can be damaged.</li>
	<li>For antigen retrieval, place slides in a metal slide holder and insert it in the beaker with boiling citrate without the aluminum foil. Let it boil for 10 min.</li>
	<li>Remove the beaker from the hot plate and let it cool for 20 min. Make sure that the adrenal sections are still covered with the buffer.</li>
	<li>Prepare the blocking solution. If using the commercially available staining kit employed for the Representative Results (see the Table of Materials), in a tube add 1.25 mL of 1x PBS, 1 drop (equal to about 45 &#181;L) of Mouse Ig Blocking Reagent, and 5% of normal goat serum. Store the blocking solution at 4 &#176;C or on ice while working. 
	NOTE: The serum used depends on the secondary antibody&#39;s host species. Here secondary antibodies raised in goat have been used. For other antibodies, different serum concentrations may be necessary. Empirically determine the correct serum concentration by testing several concentrations.</li>
	<li>Transfer the slides into a Coplin jar containing 1x PBS and wash 3x 10 min each on a rocker with gentle rocking.</li>
	<li>Prepare a humidified chamber for the staining.</li>
	<li>Gently, shake off the PBS from one slide; wipe the slide bottom with a lint-free wipe to remove the excess PBS.</li>
	<li>Using a special marker for slides with hydrophobic properties, draw a circle surrounding each adrenal section, making sure that the glass surface is dry to avoid spreading of the ink solution to the section. If necessary, carefully wipe dry the surface. Place the slide in the humidified chamber.</li>
	<li>Quickly, pipette 50 &#181;L (or enough to cover sections) of blocking solution onto every section. Incubate for 1 h at room temperature. 
	NOTE: Blocking solution volume needed may vary; it is important that the sections are well covered with the solution.</li>
	<li>Prepare the diluent solution from the commercially available kit with 7.5 mL of 1x PBS, 600 &#181;L of protein concentrate stock solution and 5% normal goat serum (or any other serum and concentration used in the blocking solution). Store the diluent solution at 4 &#176;C or on ice while working.</li>
	<li>Prepare the primary antibody solutions diluting the primary antibodies at the appropriate concentrations in the diluent solution. For co-staining, add an aliquot of each antibody in a new tube, and mix gently. Place antibodies and antibody solutions on ice until ready to use. 
	NOTE: Here we employed anti-SF1 antibody raised in rabbit and an anti-TH antibody raised in mouse. To prepare the antibody working solutions follow as directed.
	<ol>
		<li>For SF1 working solution, in one tube, add 1 &#181;L of anti-SF1 antibody to 999 &#181;L of diluent solution (final concentration of 1.5 &#181;g/mL). For TH working solution, in a tube, add 1 &#181;L anti-TH antibody in 499 &#181;L of diluent solution (estimated final concentration of 2 - 6 &#181;g/mL).</li>
		<li>For SF1 + TH antibody working mix, in a fresh tube, pipette 250 &#181;L of SF1 working solution and 250 &#181;L of TH working solution. Mix gently by pipetting. Store on ice.</li>
	</ol>
	</li>
	<li>Transfer the slides in a Coplin jar containing 1x PBS and wash 3x for 5 min each on a rocker with gentle rocking.</li>
	<li>Place the slides back into the humidified chamber after wiping off excess of PBS, pipette the SF1 + TH antibody working mix (or other primary antibody solution) on each section, close the humidified chamber and leave it overnight at 4 &#176;C.</li>
	<li>On the following day, move the slides into a Coplin jar containing 1x PBS and wash 3x for 15 min on a rocker with gentle rocking.</li>
	<li>While washing the slides, prepare the secondary antibody solution in the diluent solution using the appropriate concentration. If performing co-staining, add equal amount of each secondary antibody solution in a new tube. Mix gently by pipetting. Store on ice. Protect the tubes from light and store on ice until ready to use. 
	NOTE: Here, the secondary antibodies used are 488 nm fluorescent dye-labeled anti-mouse raised in goat and 549 fluorescent dye-labeled anti-rabbit raised in goat. The secondary antibody solution was prepared by adding 0.5 &#181;L of each secondary antibody in 799 &#181;L of diluent solution (final concentrations of 1.2 &#181;g/mL).</li>
	<li>Place the slides back in the humidified chamber after removing excess of PBS, quickly pipette the secondary antibody solution on the adrenal sections, close the humidified chamber and protect from light. Incubate for 1 h at room temperature.</li>
	<li>Wash the slides in a Coplin jar with 1x PBS and wash 3x for 15 min on a rocker with gentle rocking, making sure to protect them from light.</li>
	<li>Prepare the 4&#39;,6-diamidino-2-phenylindole (DAPI) solution for nuclear staining: 1 &#181;L of DAPI (20 mg/mL) in 1 mL of 1x PBS. 
	NOTE: Blue-fluorescent nuclear counterstain can also be achieved using Hoechst dye.</li>
	<li>Pipette DAPI solution onto the adrenal sections, cover from light and incubate at room temperature for 7 min.</li>
	<li>Wash the slides in a Coplin jar with 1x PBS and wash 3x for 5 min on a rocker with gentle rocking while protecting the slides from light.</li>
	<li>In an area shielded from direct light, lay down a cover glass, and pipette 60 &#181;L or about 3 drops of mounting agent suitable for immunofluorescence along the surface of the cover glass.</li>
	<li>Gently wipe the back of a slide with a lint-free wipe, position the slide face down toward the cover glass and parallel to it, so the tissue sections face the cover glass with the mounting agent on it. Lightly press the slide on the cover glass and make sure that no air bubble is trapped between the slide and the cover glass.
	<ol>
		<li>If necessary, apply further pressure to remove the air bubbles and the excess of mounting agent.</li>
	</ol>
	</li>
	<li>Lay down the slide in a dark container and cure at room temperature for a minimum of 24 h (or the time indicated in the information sheet of the mounting agent used) and then proceed to imaging.</li>
</ol>
7. Imaging
NOTE: A fluorescence microscope connected to a camera is required for detecting and capturing the fluorescence emitted by the tissues after excitation at determined wavelengths. While obvious, it is important to remember to choose the secondary antibody conjugated with fluorochromes whose excitation and emission spectra are compatible with the equipment available. Imaging settings vary according to the microscope and the software used for capturing images. There are some basic rules that apply for imaging adrenal sections, such as making sure that the exposure time, camera gain settings, and light source intensity are kept constant.
<ol>
	<li>Turn on the light source and the camera, launch the imaging software following the manufacturer&#39;s directions. 
	NOTE: The order of these actions can differ depending on the equipment used.</li>
	<li>Secure the slide on the stage, open the shutter, and looking into the microscope eyepieces use nuclear staining (DAPI) channel and low magnification (4X) to identify the tissue on the slide: adrenal sections are small, and their visualization may require some time.</li>
	<li>Change to a higher magnification (10X), focus, and close the shutter. 
	NOTE: It is important to avoid unnecessarily long exposition under the light that can cause photobleaching, resulting in loss of signal.</li>
	<li>Using the software commands, select the objective (10X) and the channel in use (DAPI), adjust exposure time (1 s, can be adjusted if the signal is too strong or weak). If available, set binning for live imaging (not acquisition) to &#39;2 X 2&#39;, conversion gain to &#39;mid&#39;, readout speed to &#39;20 MHz&#39;. If the software used does not display the scale bar at the end of the imaging, select the scale bar option from the menu and position the bar where desired.</li>
	<li>Open the shutter, re-adjust the focus if necessary, and switch channels (FITC, TRITC, DAPI). If the microscope is not automated, take a picture of the adrenal in each channel without moving the stage and make sure to adjust the selection of the channel in use. Close the shutter once finished. 
	NOTE: Write down the settings employed in case the software does not automatically save them.</li>
	<li>Save the pictures. 
	NOTE: Merged pictures can be often created using the microscope&#39;s software or other graphics editor software packages.</li>
	<li>Repeat imaging for other areas of the section and/or with a higher magnification. 
	NOTE: A 20X magnification often requires a shorter exposure time (i.e., 800 ms).</li>
	<li>At the end of imaging, shut off all the equipment in the proper order.</li>
</ol>

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The authors have nothing to disclose.

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.

<table><tbody><tr><td>24-well cell culture plate</td><td>Nest Biotechnology Co.</td><td>0412B</td><td></td></tr><tr><td>Disposable needles 25 G x 5/8&quot;</td><td>Exel International</td><td>26403</td><td></td></tr><tr><td>Paraformaldehyde (PFA)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P6148</td><td></td></tr><tr><td>Paraplast plus&amp;nbsp;</td><td>McCormik scientific</td><td>39502004</td><td>Paraffin for tissue embedding</td></tr><tr><td>Shandon biopsy cassettes II with attached lid</td><td>Thermo scientific</td><td>1001097</td><td>Cassettes for tissue processing</td></tr><tr><td>High Profile Microtome Blades</td><td>Accu-Edge</td><td>4685</td><td>Disposable stainless steel blades</td></tr><tr><td>Peel-a-way disposable plastic tissue embedding molds</td><td>Polysciences Inc.</td><td>18986</td><td>Truncated, 22 mm square top tapered to 12 mm bottom</td></tr><tr><td>Superfrost Plus Microscope Slides</td><td>Fisherbrand</td><td>12-550-15</td><td>75 mm x 25 mm x 1 mm</td></tr><tr><td>Xylene</td><td>Fisher Chemical</td><td>X5P1GAL&amp;nbsp;</td><td></td></tr><tr><td>200 Proof Ethanol</td><td>Decon Labs, Inc.</td><td></td><td></td></tr><tr><td>Certi-Pad Gauze pads&amp;nbsp;</td><td>Certified Safety Mfg, Inc</td><td>231-210</td><td>3&quot; x 3. Sterile latex free gauze pads</td></tr><tr><td>M.O.M kit&amp;nbsp;</td><td>Vector laboratories</td><td>BMK-2202</td><td>For detecting mouse primary antibodies on mouse tissue</td></tr><tr><td>KimWipes</td><td>Kimtech</td><td>34155</td><td>Wipes 4.4 inch x 8.4 inch</td></tr><tr><td>Super PAP PEN</td><td>Invitrogen&amp;nbsp;</td><td>00-8899</td><td>Pen to draw on slides</td></tr><tr><td>Microscope cover glass&amp;nbsp;</td><td>Fisherbrand</td><td>12-544-D</td><td>Size: 22 x 50 x 1.5</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542&amp;nbsp;</td><td>(Prepared in 20 mg/mL stock)</td></tr><tr><td>ProLong Gold antifade reagent</td><td>Molecular Probes</td><td>P36930</td><td>Mounting agent for immunofluorescence</td></tr><tr><td>X-cite series 120Q</td><td>Lumen Dynamics</td><td></td><td>Light source</td></tr><tr><td>Coolsnap Myo</td><td>Photometrics</td><td></td><td>Camera</td></tr><tr><td>Optiphot-2&amp;nbsp;</td><td>Nikon</td><td></td><td>Microscope</td></tr><tr><td>microtome</td><td>American Optical</td><td></td><td></td></tr><tr><td>Tissue embedder</td><td>Leica</td><td>EG1150 H&amp;nbsp;</td><td></td></tr><tr><td>Tissue processor&amp;nbsp;</td><td>Leica</td><td>ASP300S</td><td></td></tr><tr><td>Normal goat serum</td><td>Sigma</td><td>G9023</td><td></td></tr><tr><td>Mouse anti-TH</td><td>Millipore</td><td>MAB318</td><td>Primary antibody</td></tr><tr><td>Rabbit anti-SF1</td><td>Ab proteintech group (PTGlabs)</td><td>&amp;nbsp;custom made</td><td>Primary antibody</td></tr><tr><td>Alexa-488 Mouse IgG&amp;nbsp; raised goat</td><td>Jackson ImmunoResearch</td><td>115-545-003&amp;nbsp;</td><td>Secondary antibody</td></tr><tr><td>Dylight-549 Rabbit IgG raised goat</td><td>Jackson ImmunoResearch</td><td>111-505-003</td><td>Secondary antibody</td></tr><tr><td>Citrate acid anhydrous</td><td>Fisher Chemical</td><td>A940-500</td><td></td></tr><tr><td>NIS-Elements Basic Research&amp;nbsp;</td><td>Nikon</td><td></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...

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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 ...

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23.8K Views.  University of Michigan Health System. Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Video: Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

Biology

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Developmental Biology

Highly Resolved Intravital Striped-illumination Microscopy of Germinal Centers

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

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

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

Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals &#8722; Using Unconjugated Primary Antibodies from the Same Host Species

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling using multiple primary antibodies. To minimize antibody cross-reactivity, multiplex immunostaining using indirect staining requires unlabeled primary antibodies from different host species. However, the appropriate combination of different species antibodies is not always available. Here, we describe a method of using unlabeled primary antibodies from the same host species (e.g., in this case both antibodies are from rabbit) for multiplex immunofluorescence on formalin-fixed paraffin-embedded (FFPE) mouse adrenal sections. This method uses the same procedure and reagents used in the antigen retrieval step to strip the activity of the previously stained primary antibody complex. Slides were stained with the first primary antibody using a general immunostaining protocol followed by a binding step with a biotinylated secondary antibody. Then, an avidin-biotin-peroxidase signal development method was used with fluorophore-tyramide as the substrate. The immunoactivity of the first primary antibody complex was stripped through immersion in a microwaved boiling sodium citrate solution for 8 min. The insoluble fluorophore-tyramide deposition remained on the sample, which allowed the slide to be stained with other primary antibodies. Although this method eliminates most false positive signals, some background from antibody cross-reactivity may remain. If the samples are enriched with endogenous biotin, a peroxidase-conjugated secondary antibody may be used to replace the biotinylated secondary antibody to avoid the false positive from recovered endogenous biotin.

Several different methods have been established for multiplex immunostaining using primary antibodies from the same host species. Here, we describe the use of microwave-mediated antibody stripping and of fluorophore-tyramide to block antibody cross-reactivity during multiplex immunostaining on formalin-fixed paraffin-embedded mouse adrenal sections.

Immunostaining is widely used in biomedical research to show the cellular expression pattern of a given protein. Multiplex immunostaining allows labeling ...

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

Neuroscience

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

Indirect Immunofluorescence on Frozen Sections of Mouse Mammary Gland

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple method for the immune detection of proteins, the mouse lactating mammary gland being taken as an example. A protocol for the preparation of the tissue samples, especially concerning the dissection of mouse mammary gland, tissue fixation and frozen tissue sectioning, are detailed. A standard protocol to perform indirect immunofluorescence, including an optional antigen retrieval step, is also presented. The observation of the labeled tissue sections as well as image acquisition and post-treatments are also stated. This procedure gives a full overview, from the collection of animal tissue to the cellular localization of a protein. Although this general method can be applied to other tissue samples, it should be adapted to each tissue/primary antibody couple studied.

The indirect immunofluorescence protocol described in this article allows the detection and the localization of proteins in the mouse mammary gland. A complete method is given to prepare mammary gland samples, to perform immunohistochemistry, to image the tissue sections by fluorescence microscopy, and to reconstruct images.

Indirect immunofluorescence is used to detect and locate proteins of interest in a tissue. The protocol presented here describes a complete and simple ...

Isolation of Intact, Whole Mouse Mammary Glands for Analysis of Extracellular Matrix Expression and Gland Morphology

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal architecture. Here, a small pocket below the skin was created using Mayo scissors, allowing separation of the glands within the subcutaneous tissue from the underlying peritoneum. Visualization of the glands was aided by the use of 3.5x-R surgical micro loupes. The pelt was inverted and pinned back allowing identification of the intact mammary fat pads. Each of the #4 abdominal glands was bluntly dissected by sliding the scalpel blade laterally between the subcutaneous layer and the glands. Immediately post-harvest, glands were placed in 10% neutral buffered formalin for subsequent tissue processing. Excision of the entire gland is advantageous because it primarily eliminates the risk of excluding important tissue-wide interactions between ductal epithelial cells and other microenvironmental cellular populations that could be missed in a partial biopsy. One drawback of the methodology is the use of serial sections from fixed tissues which limits analyses of ductal morphogenesis and protein expression to discrete locations within the gland. As such, changes in ductal architecture and protein expression in 3 dimensions (3D) is not readily obtainable. Overall, the technique is applicable to studies requiring whole intact murine mammary glands for downstream investigations such as developmental ductal morphogenesis or breast cancer.

Here, we present a protocol for the isolation of whole, intact mouse mammary glands to investigate extracellular matrix (ECM) expression and ductal morphology. Mouse #4 abdominal glands were extracted from 8-10 week old female nulliparous mice, fixed in neutral buffered formalin, sectioned and stained using immunohistochemistry for ECM proteins.

The goal of this procedure was to harvest the #4 abdominal mammary glands from female nulliparous mice in order to assess ECM expression and ductal ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

A Multiplex Immunostaining Method Using Primary Antibodies from the Same Host Species

Source: Lyu, Q., et al. <a href="https://app.jove.com/v/60868">Microwaving and Fluorophore-Tyramide for Multiplex Immunostaining on Mouse Adrenals &#8722; Using Unconjugated Primary Antibodies from the Same Host Species.</a> J. Vis. Exp. (2020).
This video demonstrates an assay to perform multiplex immunostaining using primary antibodies from the same host species. In this method, an adrenal tissue section undergoes a two-step immunostaining process. Initially, the section is stained with antibodies specific for markers in the cortex, followed by stripping the bound antibody and restaining with antibodies specific for markers in the medulla.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Staining with the First Antibody

...