Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers

Summary

This protocol uses three-dimensional (3D) imaging and analysis techniques to visualize and quantify nerve-specific mitochondria. The techniques are applicable to other situations where one fluorescent signal is used to isolate a subset of data from another fluorescent signal.

Abstract

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to isolate nerve-specific mitochondria and evaluate disease-induced alterations of mitochondria in the distal tip of sensory nerves. The protocol combines fluorescence immunohistochemistry, confocal microscopy and 3D image analysis techniques to visualize and quantify nerve-specific mitochondria. Detailed parameters are defined throughout the procedures in order to provide a concrete example of how to use these techniques to isolate nerve-specific mitochondria. Antibodies were used to label nerve and mitochondrial signals within tissue sections of skin punch biopsies, which was followed by indirect immunofluorescence to visualize nerves and mitochondria with a green and red fluorescent signal respectively. Z-series images were acquired with confocal microscopy and 3D analysis software was used to process and analyze the signals. It is not necessary to follow the exact parameters described within, but it is important to be consistent with the ones chosen throughout the staining, acquisition and analysis steps. The strength of this protocol is that it is applicable to a wide variety of circumstances where one fluorescent signal is used to isolate other signals that would otherwise be impossible to study alone.

Introduction

Mitochondria serve vital cellular functions that include producing cell energy, buffering calcium, and regulating necrotic and apoptotic cell death^1,2,3. The nervous system has a high metabolic rate compared to the body⁴ suggesting that neurons generate a high degree of cellular energy in the form of adenosine triphosphate (ATP) through mitochondrial respiration. A lot of evidence documents that neuronal functions are dependent on ATP⁵, especially at the synapses⁶. Therefore, the distribution of mitochond....

Protocol

Skin punch biopsies were obtained from subjects that were recruited from a large community-based primary care network at the University of Utah Diabetes Center (Salt Lake City, UT). This study was approved by the University of Michigan Institutional Review Board and complied with the tenets of the Declaration of Helsinki. Written informed consent was obtained from each subject prior to testing.

1. Fluorescence Immunohistochemistry

1. Prepare punch biopsies for intraepidermal nerve fiber immunohistochemistry:
   1. Perform 3 mm skin biopsies by medical staff and place the whole biopsy in 1.5 mL Zamboni's f....

Results

Visualization and quantification of mitochondria within human IENFs

Fluorescence immunohistochemistry allows for the simultaneous labeling of multiple signals within human skin biopsies to visualize nerves, mitochondria, and nuclei. A 96-well plate is a convenient way to organize the steps in the immunohistochemistry procedure. Figure 1 shows that this configuration accounts for up .......

Discussion

This protocol is designed to isolate, quantify and analyze the size and distribution of nerve-specific mitochondria within IENFs in 3D from human skin biopsies. There are several critical steps in the protocol. The free-floating fluorescence immunohistochemistry is designed to stain and analyze multiple signals in each sample, providing a more versatile methodology for explorative research^44,45. This procedure allows for penetration of the antibodies into the tis.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2% Zamboni's Fixative	Newcomer Supply, Middleton, WI	1459A	2% paraformaldehyde, 0.2% saturated picric acid in phosphate buffered saline (PBS), pH 7.4
10x Phosphate Buffered Saline (PBS)	Fisher Scientific, Pittsburgh, PA	BP399-4	To make up 1x PBS
Image-iT FX Signal Enhancer	ThermoFisher Scientific, Waltham, Massachusetts	I36933	enhances Alexa Fluor dye signals by reducing nonspecific binding
Anti-Protein Gene Product 9.5 Antibody (Rabbit Polyclonal)	Proteintech Group Inc. Rosemont, IL	14730-1-AP	abbreviated as PGP9.5, replaces discontinued AbD Serotec (Cat. No. 7863-0504) antibody
Anti-Pyruvate Dehydrogenase E2/E3bp Antibody (Mouse Monoclonal)	abcam, Cambridge, MA	ab110333	abbreviated as PDH
Goat anti-mouse Secondary antibody Alexa Fluor 594 conjugate	ThermoFisher Scientific, Waltham, Massachusetts	A-11034	red-fluorescent conjugated secondaryantibody
Goat anti-rabbit Secondary antibody Alexa Fluor 488 conjugate	ThermoFisher Scientific, Waltham, Massachusetts	A-11032	green-fluorescent conjugated secondaryantibody
Albumin, from Bovine Serum	Sigma-Aldrich, St. Louis, MO	A7906-100	abbreviated as BSA
Triton X- 100	Sigma-Aldrich, St. Louis, MO	T9284	abbreviated as TX-100
0.22 µm Filter	EMD Millipore, Billerica MA	MILLEX GP SLGP 033NS	0.22 µm Millipore filter
Parafilm M	Fisher Scientific, Pittsburgh, PA	13-374-10	Curwood Wisconsin LLC Parafilm M (PM-996)
Non-calibrated Loop	Fisher Scientific, Pittsburgh, PA	22-032092	inoculating Loop by Decon LeLoop (MP 199-25)
96-well Assay Plate	Corning Incorporated, Corning, NY	3603	96-well flat bottom plate
Prolong Gold antifade reagent with DAPI	ThermoFisher Scientific, Waltham, Massachusetts	P-36931	DAPI staining of nuclei
Microscope Cover Glass 50 x 24 mm	Fisher Scientific, Pittsburgh, PA	12-544E	Coverslips
Superfrost Plus Microscope Slides	Fisher Scientific, Pittsburgh, PA	12-550-15	Microscope Slides
Leica SP5 Laser Scanning Confocal Microscope	Leica Microsystems, Buffalo Grove, IL	SP5	Confocal Microscope
Volocity x64 Software	Perkin Elmer, Waltham , MA	version 4.4.0	Volocity software is used for Steps 3.1 and 3.2 in the protocol for image processing
Imaris x64 3 Dimensional Analysis Software	Bitplane, Concord, MA	version 7.7.1	Imaris software is used for Steps 3.3 through 3.5 in the protocol for image analysis
Excel	Microsoft, Redmond, WA	version Office 2013	Excel spreadsheet software is used for Step 3.6 in the protocol to summarize morphometric features
Optimum Cutting Temperature Compound	Sakura Finetek USA, Inc., Torrance, CA	4583	abbreviated as OCT
Leica Cryostat	Leica Biosystems, Buffalo Grove, IL	CM1850	Cryostat for cutting 50 µm sections
CellLight Mitochondria-GFP, BacMam 2.0	ThermoFisher Scientific, Waltham, Massachusetts	C10600	Used as a postive control to label mitochondria with a green fluorescent signal