Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

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.......

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.......

Materials

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