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Immunology and Infection

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Published: October 2nd, 2018

DOI:

10.3791/58530

1Department of Internal Medicine (Division of Metabolism, Endocrinology, and Diabetes), University of Michigan Health System, 2Department of Cell and Developmental Biology, University of Michigan Health System, 3Department of Molecular and Integrative Physiology, University of Michigan Health System, 4Endocrine Oncology Program, University of Michigan Health System, 5Comprehensive Cancer Center, 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.

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 ....

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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

  1. 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 °C.
    NOTE: 4% PFA is not stable for more than 48 h.
    CAUTION: PFA is toxic, avoid contact with ski.......

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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 an.......

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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.......

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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).

....

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Name Company Catalog Number Comments
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 4.4x8.4 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

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