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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Abstract

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.

Introduction

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 ....

Protocol

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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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).

....

Materials

NameCompanyCatalog NumberComments
24-well cell culture plateNest Biotechnology Co.0412B
Disposable needles 25Gx5/8"Exel International26403
Paraformaldehyde (PFA)Sigma-Aldrich P6148
Paraplast plus McCormik scientific39502004Paraffin for tissue embedding
Shandon biopsy cassettes II with attached lidThermo scientific1001097Cassettes for tissue processing
High Profile Microtome BladesAccu-Edge4685Disposable stainless steel blades
Peel-a-way disposable plastic tissue embedding moldsPolysciences Inc.18986Truncated,22mm square top tapered to 12mm bottom
Superfrost Plus Microscope SlidesFisherbrand12-550-1575x25x1 mm
XyleneFisher ChemicalX5P1GAL 
200 Proof EthanolDecon Labs, Inc.
Certi-Pad Gauze pads Certified Safety Mfg, Inc231-2103"x3. Sterile latex free gauze pads
M.O.M kit Vector laboratoriesBMK-2202For detecting mouse primary antibodies on mouse tissue
KimWipesKimtech34155Wipes 4.4x8.4 inch
Super PAP PENInvitrogen 00-8899Pen to draw on slides
Microscope cover glass Fisherbrand12-544-DSize: 22x50x1.5
DAPISigmaD9542 (Prepared in 20mg/mL stock)
ProLong Gold antifade reagentMolecular ProbesP36930Mounting agent for immunofluorescence
X-cite series 120QLumen DynamicsLight source
Coolsnap MyoPhotometricsCamera
Optiphot-2 NikonMicroscope
microtomeAmerical Optical
Tissue embedderLeicaEG1150 H 
Tissue processor LeicaASP300S
Normal goat serumSigmaG9023
Mouse anti-THMilliporeMAB318Primary antibody
Rabbit anti-SF1Ab proteintech group (PTGlabs) custom madePrimary antibody
Alexa-488 Mouse IgG  raised goatJackson ImmunoResearch115-545-003 Secondary antibody
Dylight-549 Rabbit IgG raised goatJackson ImmunoResearch111-505-003Secondary antibody
Citrate acid anhydrousFisher ChemicalA940-500
NIS-Elements Basic Research NikonSoftware for imaging

References

  1. Brandtzaeg, P. The increasing power of immunohistochemistry and immunocytochemistry. Journal of Immunological Methods. 216 (1-2), 49-67 (1998).
  2. Matos, L. L., Trufelli, D. C., de Matos, M. G., da Silva Pinhal, M. A.

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ImmunofluorescenceAdrenal GlandMouseIsolationFixationTissue ProcessingParaffin EmbeddingMicrotome Sectioning

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