Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

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 females^6,7. 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 unknown^8,9.

Cells of the adrenal cortex contain lipid droplets that store cholesterol esters that serve as the precursor of all steroid hormones^10,11.  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.