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 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.
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 "original" 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. The term "steroidogenesis" 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 "chromaffin" 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.
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
2. Adrenal Dissection
3. Peri-adrenal Fat Removal
4. Tissue Processing and Embedding
5. Sectioning with a Rotary Microtome
6. Immunofluorescence
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.
Figure 1 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 and the sections are imaged at the microscope.
The removal of adjacent fat (Figure 2A) is important for facilitating further processing and sectioning of the adrenal glands. Successful removal of the surrounding adipose tissue is shown in Figure 2B, where the adrenal is easily detectable and no extra fat is visible. During this step it is critical, however, to not let the adrenal gland dry out or this could damage the tissue structure. Dryness is recognizable when the adrenal assumes a wrinkly appearance (Figure 2C). This issue can be easily overcome by rehydrating the adrenal in 1x PBS before continuing with the fat removal.
The end results obtained following this protocol are presented in Figure 3. The immunofluorescence images illustrate the immunostaining of an adrenal gland at two different magnifications. Red nuclear SF1 staining labels the adrenocortical cells, whereas cytoplasmic green staining labels cells of the medulla. The outer capsule is labeled by nuclear staining in blue (DAPI) since it is not steroidogenic (SF1-negative).
Figure 1: Schematic representation of the protocol. After harvesting the adrenal glands and removing the adjacent adipose tissue, the tissues are fixed in 4% PFA, processed for paraffin embedding, and sectioned. The sections are then immunostained and imaged with a fluorescence microscope. Please click here to view a larger version of this figure.
Figure 2: Removal of peri-adrenal fat. (A) The fat surrounding the adrenal gland is removed under a dissection microscope. (B) Adrenal gland after the clean up. (C) Example of tissue drying up and that requires hydration. Please click here to view a larger version of this figure.
Figure 3: Immunofluorescence imaging of the adrenal gland. Example of immunofluorescence imaging (at different magnifications) of an adrenal gland stained with markers of the adrenal cortex (SF1, nuclear staining) and of the adrenal medulla (TH, cytoplasmic green). Nuclei (DAPI) are labeled in blue. C: capsule; CO: cortex; m: medulla. Scale bars = 200 µm. Please click here to view a larger version of this figure.
Reagent | Station | Temperature | Duration |
70% EtOH | 1 | RT | 1 h |
90% EtOH | 2 | RT | 1 h |
90% EtOH | 3 | RT | 1 h |
Absolute EtOH | 4 | RT | 1 h |
Absolute EtOH | 5 | RT | 1 h |
Absolute EtOH | 6 | RT | 1 h |
Absolute EtOH | 7 | RT | 1 h |
Xylene | 8 | RT | 1 h |
Xylene | 9 | RT | 1 h |
Xylene | 10 | RT | 1 h |
Paraffin wax | 11 | 62 °C | 1 h |
Paraffin wax | 12 | 62 °C | 1 h |
Paraffin wax | 13 | 62 °C | 1 h |
Table 1: Tissue processor program.
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 laboratory, the incubation in 4% PFA can vary from 1 h to 4 h, while in other laboratories the fixation time is extended to 12–24 h16. In our hands, however, a longer fixation time led to increased background noise and was not optimal for several antibodies routinely used in our research.
The pH of antigen retrieval can also play a role in the success of staining. Heat-induced epitope retrieval (HIER) can be carried out by employing commercially available solutions with pH ranging from acidic to alkaline, as well as other in-house made buffers such as citrate (pH 6), ethylenediaminetetraacetic acid (EDTA, pH 8), Tris-EDTA (pH 9), Tris (pH 10). Enzymatic treatment can also be an option for antigen unmasking. Incubating the deparaffinized slides for a limited time with a solution containing a proper concentration of an enzyme (for example proteinase K) can be an alternative method to HIER. It is, however, essential to titrate the enzyme concentration and to determine the ideal incubation time, since over-digestion can negatively impact the tissue17.
The choice of blocking buffer is also another variable for troubleshooting. Besides the use of normal goat serum and the commercially available blocking buffer mentioned in this protocol (convenient in this instance when using primary mouse antibodies on mouse tissues to prevent high background due to endogenous mouse IgG), there are additional options available such as protein solutions (bovine serum albumin (BSA) or dry milk) or other commercially available buffers. It is critical to make sure that the buffer does not contain substances that can interfere with the staining, such as biotin when using a biotin-streptavidin based method.
The adrenal gland is an endocrine organ characterized by high lipid content that can often make immunostaining challenging due to high autofluorescence of the lipid. Moreover, adrenal cortices from mice and rats are rich in autofluorescent intracytoplasmic lipofuscin, a pigmented granular and amorphous material that ranges in color from yellow to brown. To overcome this problem, compounds such as Sudan Black B (SBB) can quench the fluorescence generated by lipofuscins18. Another factor that compromises the outcome of a good staining is the presence of blood cells. Hemoglobin present in the erythrocytes absorbs light of wavelengths <600 nm and can interfere with fluorochromes that span that wavelength19,20. While perfusion techniques can be used to clear blood cells from tissue vessels, the use of 10 mM copper sulfate at pH 5 before performing nuclear counterstaining can also help in suppressing the unwanted fluorescence21.
A critical step in this protocol is the adrenal dissection. The adrenal gland is an organ whose location can be difficult to locate in situ: in mice, the glands are small and their position is somewhat variable. Especially in older animals, adrenals are also surrounded by adipose tissue that can interfere with the detection of the glands and their consequent isolation, for this reason it is crucial to have a clear view of the area during this step of the protocol. Removal of peri-adrenal fat removal is a delicate step. Particular attention should be paid to the adrenals while detaching the fat so as not to rupture the capsule. The gland must also be kept moist with PBS during the procedure to avoid tissue damage.
When sectioning, detecting the presence of the small portion of adrenal tissue in the sections can be challenging due to tissue hypopigmentation after the processing step. A microscope is very helpful during the sectioning for discerning the tissue from the wax.
Immunostaining on paraffin-embedded sections is a valuable technique for immunolabeling proteins of interest while preserving the morphology of the tissue. The method presented here is based upon fluorescence detection and, while it is particularly functional for studies employing multiple primary antibodies, the fluorescence signal is light-sensitive and can be easily lost or weakened if the slides are not handled properly (i.e., prolonged exposure to the microscope light or unnecessary exposure to ambient light). Moreover, we can notice a decline of the quality of the fluorescence signal itself over time. This problem can be avoided by using chromogenic detection, which is photostable and can be visualized for many years. This method, however, lacks the advantages of the fluorescence detection, such as higher labeling precision and simultaneous labeling of several proteins in one study.
Paraffin embedding is a convenient method to process and store multiple tissue samples. However, the tissue processing for paraffin embedding itself is not suitable for imaging of fluorescent reporters endogenously expressed in some transgenic animals without the use of a specific antibody targeting the reporter. Cryopreservation is a method to avoid degradation of the fluorescent protein and allow its direct visualization under a microscope. Avoiding the use of an extra antibody can represent an advantage. On the other hand, cryopreservation can also be limiting since it affects tissue morphology; it also requires different equipment for sectioning, and the storage of slides and tissue blocks is possible in freezers only.
One major limitation of imaging a sectioned tissue is the ability to obtain images of structural components at high spatial resolutions. Tissue composition, in fact, is a major variable in determining the quality of the imaging since it influences light penetration and can lead to poor resolution. The adrenal gland is a tissue rich in lipids that cause light scattering22. In the brain, which also has a high lipid content, tissue-clearing techniques such as CLARITY, BABB, iDISCO, and 3DISCO have been developed to improve tissue visualization, allowing for better imaging and 3D tissue reconstruction23. These techniques are providing researchers high quality imaging data, and are being adapted to a various range of tissues, and, in the future, we hope to employ these methods for adrenal imaging as well.
The authors have nothing to disclose.
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).
24-well cell culture plate | Nest Biotechnology Co. | 0412B | |
Disposable needles 25Gx5/8" | Exel International | 26403 | |
Paraformaldehyde (PFA) | Sigma-Aldrich | P6148 | |
Paraplast plus | McCormik scientific | 39502004 | Paraffin for tissue embedding |
Shandon biopsy cassettes II with attached lid | Thermo scientific | 1001097 | Cassettes for tissue processing |
High Profile Microtome Blades | Accu-Edge | 4685 | Disposable stainless steel blades |
Peel-a-way disposable plastic tissue embedding molds | Polysciences Inc. | 18986 | Truncated,22mm square top tapered to 12mm bottom |
Superfrost Plus Microscope Slides | Fisherbrand | 12-550-15 | 75x25x1 mm |
Xylene | Fisher Chemical | X5P1GAL | |
200 Proof Ethanol | Decon Labs, Inc. | ||
Certi-Pad Gauze pads | Certified Safety Mfg, Inc | 231-210 | 3"x3. Sterile latex free gauze pads |
M.O.M kit | Vector laboratories | BMK-2202 | For detecting mouse primary antibodies on mouse tissue |
KimWipes | Kimtech | 34155 | Wipes 4.4×8.4 inch |
Super PAP PEN | Invitrogen | 00-8899 | Pen to draw on slides |
Microscope cover glass | Fisherbrand | 12-544-D | Size: 22x50x1.5 |
DAPI | Sigma | D9542 | (Prepared in 20mg/mL stock) |
ProLong Gold antifade reagent | Molecular Probes | P36930 | Mounting agent for immunofluorescence |
X-cite series 120Q | Lumen Dynamics | Light source | |
Coolsnap Myo | Photometrics | Camera | |
Optiphot-2 | Nikon | Microscope | |
microtome | Americal Optical | ||
Tissue embedder | Leica | EG1150 H | |
Tissue processor | Leica | ASP300S | |
Normal goat serum | Sigma | G9023 | |
Mouse anti-TH | Millipore | MAB318 | Primary antibody |
Rabbit anti-SF1 | Ab proteintech group (PTGlabs) | custom made | Primary antibody |
Alexa-488 Mouse IgG raised goat | Jackson ImmunoResearch | 115-545-003 | Secondary antibody |
Dylight-549 Rabbit IgG raised goat | Jackson ImmunoResearch | 111-505-003 | Secondary antibody |
Citrate acid anhydrous | Fisher Chemical | A940-500 | |
NIS-Elements Basic Research | Nikon | Software for imaging |