Name: Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers (Video) | JoVE Journal
Uploaded: 2017-09-29T12:30:00.000+00:00
Duration: 10 min 31 s
Description: Learn three-dimensional imaging and analysis of mitochondria within human intraepidermal nerve fibers using fluorescence immunohistochemistry. This protocol enables researchers to isolate nerve-specific signals and study mitochondrial function in neurological disease.

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This work was supported by National Institutes of Health Grants K08 NS061039-01A2, The Program for Neurology Research &#38; Discovery, and The A. Alfred Taubman Medical Research Institute at the University of Michigan. This work used the Morphology and Image Analysis Core of the Michigan Diabetes Research Center, funded by National Institutes of Health Grant 5P90 DK-20572 from the National Institute of Diabetes and Digestive and Kidney Diseases. The authors would like to thank J. Robinson Singleton and A. Gordon Smith (University of Utah) for their generous donation of human skin samples.

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
<ol>
	<li>Prepare punch biopsies for intraepidermal nerve fiber immunohistochemistry:
	<ol>
		<li>Perform 3 mm skin biopsies by medical staff and place the whole biopsy in 1.5 mL Zamboni&#39;s fixative solution (2% paraformaldehyde, 0.3% saturated picric acid in phosphate buffered saline (PBS), pH 7.4) at 4 &#176;C overnight.</li>
		<li>Rinse samples with a solution of 30% sucrose in PBS at 4 &#176;C for 16-24 h or until the sample sinks.</li>
		<li>Embed samples in optimal cutting temperature compound (OCT) using a cryomold. Place the whole 3 mm biopsy with the epidermis facing down in the mold and fill the mold with approximately 2 mL of OCT. Freeze the mold on crushed dry ice. Store at -80 &#176;C until ready to use.</li>
		<li>Cut 50 &#181;m thick cross sections using a cryostat and store in individual wells of a 96-well plate using 180 &#181;L antifreeze storage solution per well (30% ethylene glycol, 30% glycerol in PBS). The following directions are for 8 wells of a 96 well plate. Stain sections from each biopsy site that are 200 - 300 &#181;m away from each other.</li>
	</ol>
	</li>
</ol>
DAY 1:
<ol start="2">
	<li>Quench non-specific labeling of the stratum corneum:
	<ol>
		<li>Label 96-well plate as shown in Figure 1.</li>
		<li>Pipet 150 &#181;L of stock signal enhancer solution to reduce nonspecific binding of secondary antibodies34,35 into each well. Transfer sections into signal enhancer solution using an inoculating loop. 
		NOTE: Take care while working with the tissue to avoid damaging or tearing the tissue sections. Keep in signal enhancer solution for 30 minutes at room temperature on a flat rocker.</li>
		<li>Prepare rinse wells in rows 2 and 3 of the 96-well plate by adding 150 &#181;L of 1x phosphate buffered saline (PBS) into each well.</li>
		<li>Carefully transfer sections into row 2 and rinse in 1x PBS for 10 min at room temperature.</li>
		<li>Rinse a second time in 1x PBS for 10 min at room temperature (row 3).</li>
	</ol>
	</li>
	<li>Prepare 5% bovine serum albumin (BSA) blocking solution:
	<ol>
		<li>Prepare 5% blocking solution that contains 5% BSA and 0.3% Triton X 100 (TX-100) in 0.1 M PBS (see Table 1) while sections are incubating in signal enhancer solution. BSA does not go into solution easily. Vortex solution until BSA completely dissolves.</li>
		<li>Prepare blocking wells in row 4 of the 96-well plate by adding 150 &#181;L of 5% BSA blocking solution into each well.</li>
		<li>Transfer sections into individual wells of 5% BSA blocking solution and incubate sections in 5% BSA blocking solution for 1 - 2 h at room temperature on a flat rocker.</li>
	</ol>
	</li>
	<li>Prepare 1% BSA rinsing solution and dilution of primary antibodies:
	<ol>
		<li>Prepare 1% rinsing solution that contains 1% BSA and 0.3% TX-100 in 0.1 M PBS (see Table 2) while sections are incubating in 5% BSA blocking solution. BSA does not go into solution easily. Vortex solution until BSA completely dissolves.</li>
		<li>Dilute primary antibodies in 1% BSA rinsing solution while sections are incubating in 5% BSA blocking solution.
		<ol>
			<li>Make 1,500 &#181;L of primary antibody solution and add to each well in row 5.</li>
			<li>Dilute the primary antibodies: Use the nerve-specific label, rabbit polyclonal anti-protein gene product 9.5 (PGP9.5) at 1:1,000. Use the mitochondria-specific label, mouse monoclonal anti-pyruvate dehydrogenase E2/E3bp antibody (PDH) at 1:100 in 1% BSA rinsing solution.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Prepare primary antibody:
	<ol>
		<li>Pipet 150 &#181;L of primary antibody into each well in row 5 of the 96-well plate.</li>
		<li>Using the loop tool, transfer sections from the blocking solution (row 4) into the row 5 containing the primary antibody.</li>
		<li>Wrap the plate tightly with parafilm to keep it from drying out.</li>
		<li>Place samples on flat rocker at room temperature for 1 h, and then incubate samples at 4 &#186;C on a flat rocker overnight.</li>
	</ol>
	</li>
</ol>
DAY 2:
<ol start="6">
	<li>Rinse samples:
	<ol>
		<li>Prepare rinse wells in rows 6, 7 and 8 of the 96-well plate by adding 150 &#181;L of 1% BSA rinsing solution into each well.</li>
		<li>Transfer sections into first 1% BSA rinsing solution (row 6) and incubate for 1 h at room temperature. Repeat rinses twice more by incubating sections in 1% BSA rinsing solution in rows 7 and 8 for 1 h each at room temperature.</li>
	</ol>
	</li>
	<li>Dilute secondary antibodies in 1% BSA rinsing solution:
	<ol>
		<li>Make 1,500 &#181;L of secondary antibody solution while sections are incubating in the last rinse of 1% BSA rinsing solution (row 8).</li>
		<li>Dilute secondary antibodies: for PGP9.5 (green-fluorescent conjugated goat anti-rabbit antibody, 1:1000), for PDH (red-fluorescent conjugated goat anti-mouse, 1:1,000) in 1% BSA rinsing solution.</li>
	</ol>
	</li>
	<li>Prepare secondary antibody:
	<ol>
		<li>Pipet 150 &#181;L of secondary antibody into row 9 of the 96-well plate.</li>
		<li>Gently transfer the sections from 1% BSA rinsing solution (row 8) into secondary antibody wells (row 9).</li>
		<li>Using parafilm, wrap the plate tightly to keep it from drying out. Cover with aluminum foil. Place samples on flat rocker at room temperature for 1 h, and then incubate samples at 4 &#730;C on a flat rocker overnight.</li>
	</ol>
	</li>
</ol>
DAY 3:
<ol start="9">
	<li>Prepare clean 1x PBS by filtering through a 0.22 &#181;m filter:
	<ol>
		<li>Pipet 150 &#181;L of filtered 1x PBS into row 10, 11, and 12.</li>
		<li>Transfer samples to row 10 and rinse in 1x PBS for 1 h at room temperature. Cover the 96-well plate with aluminum foil and place on a flat rocker during rinse. Repeat filtered 1x PBS rinse two more times for 1 h each at room temperature in rows 11 and 12.</li>
	</ol>
	</li>
	<li>Prepare microscope slides for mounting tissue sections:
	<ol>
		<li>Place 50 &#181;L of filtered 1x PBS on a slide.</li>
		<li>Transfer section from 1x PBS (row 12) into the 50 &#181;L drop. Carefully position the section in the drop of PBS by unfolding the tissue and gently flattening it onto the glass slide. Remove excess PBS with a glass pipette to avoid diluting the mounting reagent. Do not touch sections with the glass bulb.</li>
		<li>Pipet 1 - 2 drops of mounting reagent containing DAPI directly on top of the section on the microscope slide using care to not disturb the orientation of the section. Gently place a 50 mm x 24 mm #1.5 microscope glass coverslip over the section.</li>
		<li>Clear any air bubbles that formed while placing the coverslip and wipe excess liquid off the edges of the coverslip. Prepare a new slide and repeat the mounting process for each section.</li>
		<li>Cure/dry the mounting media by placing the slides in the dark overnight at room temperature. Transfer the slides to 4 &#176;C for short-term (1 - 2 weeks) or -20 &#176;C for long-term storage (greater than 2 weeks). 
		NOTE: Negative controls omitted primary antibodies for PGP9.5 or PDH in step 1.4.2.2 and displayed no distinguishable labeling of nerves or mitochondria in the epidermis. Positive controls were performed for the PDH antibody to prove it labels all mitochondria (data not shown). Positive controls were performed on cultured primary mouse dorsal root ganglion neurons that were transduced with a baculovirus to label mitochondria with a Green Fluorescent Protein (GFP) signal and then fixed and stained with the PDH antibody and red fluorescent secondary antibody. All mitochondria that were expressing GFP were co-labeled with the red label for PDH immunohistochemistry (data not shown).</li>
	</ol>
	</li>
</ol>
2. Confocal Imaging
<ol>
	<li>Perform confocal imaging:
	<ol>
		<li>Collect images using a laser scanning confocal microscope with a 40X oil-immersion (1.25 numerical aperture (N.A.)) objective on an inverted microscope.
		<ol>
			<li>At each focal plane sequentially acquire fluorescent signals: 
			Nuclei: excitation &#955; = 405 nm, spectral emission filter &#955; = 420 - 480 nm 
			Nerve fibers: excitation &#955;= 488 nm, spectral emission filter &#955; = 505 - 560 nm 
			Mitochondria: excitation &#955; = 543 nm, spectral emission filter &#955;= 606 - 670 nm</li>
		</ol>
		</li>
		<li>Enter the following scan parameters into the microscope software: scan rate of 600 Hz with 2 frame averaging and zoom of 2.2; 12-bit intensity resolution (4096 gray levels).
		<ol>
			<li>Set the microscope software for optimized lateral resolution (scan resolution = 1,024 x 1,024) and axial resolution/optical sectioning (confocal aperture = 1 airy unit (AU) with z-step size of 210 nm). 
			NOTE: The resulting XYZ resolution is 172.2 nm x 172.2 nm x 210 nm with an image size of 176.1 &#181;m x 176.1 &#181;m x 30-50 &#181;m.</li>
		</ol>
		</li>
		<li>Activate a live scan for the nerve signal (green-fluorescence) and adjust the z-focus control to find and set the upper and lower focal planes in the microscope software that encompass the nerve signal within the tissue section. The total z-range is typically 30-50 &#181;m for a 50 &#181;m tissue section.</li>
		<li>Rotate the scan field with the microscope software during a live scan so that the epidermis is horizontally or vertically positioned in the image.</li>
		<li>Scan each signal separately and adjust the detector (photomultiplier tube, PMT) voltage and offset to minimize/remove any over and under saturated pixels. 
		NOTE: Scan times with the above parameters take approximately 20 - 40 min depending on the number of z slices.</li>
	</ol>
	</li>
</ol>
3. 3D Visualization and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers
<ol>
	<li>Isolate the 3D epidermis:
	<ol>
		<li>Duplicate the original image and use the maximum intensity projection (extended focus view) of the image to identify and isolate the epidermis.</li>
		<li>Use a region of interest tool to trace along the upper and lower edges of the epidermis to remove unwanted areas such as the stratum corneum and dermis that are absent of intraepidermal nerve fibers. Crop to this selection.</li>
	</ol>
	</li>
	<li>Use deconvolution on the nerve and mitochondrial fluorescent signals: 
	NOTE: Deconvolution helps to restore the integrity of the fluorescent signals. The restoration used in this protocol is called blind deconvolution because it uses the fluorescent signals in the images to determine how much the signals spread from their original source (point spread function). The process improves signal resolution by reassigning the signal spread back to its origin location.
	<ol>
		<li>Calculate a point spread function (PSF) for the green fluorescent nerve signal (green-fluorescence) with the following parameters:
		<ol>
			<li>Set calculated PSF to confocal. Set medium refractive index to 1.515 and numerical aperture to 1.25. Set detector pinhole to 1 Airy unit (A.U.). Set laser excitation wavelength to 488 nm and emission wavelength to 515 nm.</li>
		</ol>
		</li>
		<li>Calculate a PSF for the red fluorescent mitochondrial signal (red-fluorescence) with the following parameters:
		<ol>
			<li>Set calculated PSF to confocal. Set medium refractive index to 1.515 and numerical aperture to 1.25. Set detector pinhole to 1 AU. Set laser excitation wavelength to 543 nm and emission wavelength to 617 nm.</li>
		</ol>
		</li>
		<li>Optimize the nerve and mitochondria fluorescent signals by deconvolution using the corresponding PSFs listed above and iterative restoration feature set at 100% confidence and an iteration limit of 10 cycles.</li>
	</ol>
	</li>
	<li>Create nerve-specific surfaces:
	<ol>
		<li>Use the &#34;create surface&#34; tool to make a solid surface of the nerves from the deconvolved green- fluorescent secondary labeling of the PGP9.5 identified nerves.</li>
		<li>Uncheck the &#34;smooth&#34; feature and use the absolute intensity feature to set the threshold for the nerve signal since it is significantly brighter than background fluorescence.</li>
		<li>Use the absolute intensity feature to set the threshold for the nerve signal since it is significantly brighter than background fluorescence. Set threshold value low enough to accurately identify the nerves.</li>
		<li>Filter out small, non-nerve surfaces based on size. 
		NOTE: If necessary, manually edit out additional non-nerve surfaces within the &#34;Edit&#34; tab by holding the CONTROL key to highlight multiple objects and then deleting them with the Delete key.</li>
	</ol>
	</li>
	<li>Isolate nerve-specific fluorescent mitochondrial signal:
	<ol>
		<li>Select the Edit tab of the nerve surface to view the &#34;Mask Properties&#34; feature. The nerve surface created in steps 3.3 is used to isolate mitochondria within those nerves away from mitochondrial signals associated with the keratinocytes.</li>
		<li>As the &#34;Mask All&#39; button opens a &#34;Mask Channel&#34; window, choose the deconvolved red-fluorescent signal from the pull down menu under &#34;Channel Selection&#34; for the mitochondrial signal.</li>
		<li>Click in the box to put a check mark in the &#34;duplicate channel before applying mask&#34; option.</li>
		<li>Click on the radio button in front of the &#34;Constant inside/outside&#34; option of the Mask Settings and click in the box to put a check mark in the &#34;Set voxel outside surface to&#34; option and type in 0.00 for the value. Click OK button to create the new channel that represents mitochondrial signals within the nerve surface.</li>
	</ol>
	</li>
	<li>Create mitochondria-specific surfaces:
	<ol>
		<li>Use the &#34;create surface&#34; tool to make a solid surface of the mitochondria from the newly created fluorescent channel of the nerve-specific mitochondrial signals from steps 3.4.</li>
		<li>Uncheck the &#34;smooth&#34; feature and select the &#34;background subtraction&#34; feature to set the threshold. This feature uses local contrast around the mitochondrial signal to identify mitochondria from background.</li>
		<li>Set threshold value low enough to accurately identify mitochondria. In this example the lower threshold was set at 2,000 for a 16-bit (65,536) scale.</li>
		<li>Filter mitochondrial surfaces based on size. In this example the voxel limit was set to 1.0 voxels, which is the lowest limit possible. If necessary, manually edit out non-mitochondria surfaces within the &#34;Edit&#34; tab by holding the CONTROL key to select multiple objects and then deleting them with the Delete key. 
		NOTE: Occasionally, the software will create surfaces that are not associated with a distinguishable fluorescent mitochondrial signal. In these cases, it is possible to remove them with the &#34;Edit&#34; tab.</li>
	</ol>
	</li>
	<li>Export and calculate morphometric values for analysis:
	<ol>
		<li>Export values for nerve and mitochondrial surfaces from the &#34;Statistics&#34; tab for further analysis with electronic spreadsheet software.</li>
		<li>Export the volume values for both the nerve and mitochondrial surfaces.</li>
		<li>Count the number of individual nerve fibers present in each image and record the value in the electronic spreadsheet.</li>
		<li>For each image, calculate the following potential values for analysis in the electronic spreadsheet:
		<ol>
			<li>Sum the volumes for all nerve surfaces.</li>
			<li>Filter out nerve-specific mitochondrial surfaces below a volume of less than 0.02 &#181;m3 and bin the surfaces by size. For example, use bins such as 0.02 - 0.04, 0.04 - 0.08, 0.08 - 0.16, 0.16 - 0.32, 0.32 - 0.64, 0.64 - 1.28, 1.28 - 2.56, greater than 2.56 &#181;m3. 
			NOTE: The lower limit of mitochondrial volume was set to 0.02 &#181;m3 based on previously published volumes of mitonchodria36,37,38.</li>
			<li>Count the number of nerve-specific mitochondrial surfaces within each bin and the total number of surfaces over the bins (0.02 - greater than 2.56 &#181;m3).</li>
			<li>Calculate the proportion of nerve-specific mitochondrial surfaces in each bin. Use the count per bin divided by the total number of mitochondria surfaces.</li>
			<li>Sum the volumes for all nerve-specific mitochondrial surfaces.</li>
			<li>Calculate the proportion of nerve with mitochondrial signal by dividing the total nerve surface volume by the sum of all mitochondrial surface volumes.</li>
			<li>Calculate the number of mitochondria per nerve volume by dividing the total nerve surface volume by the count of all mitochondrial surfaces.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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<li>Petrozzi, L., Ricci, G., Giglioli, N. J., Siciliano, G., Mancuso, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria+and+neurodegeneration%5BTitle%5D)+AND+%22Biosci+Rep%22%5BJournal%5D)">Mitochondria and neurodegeneration.</a> Biosci Rep. 27 (1-3), 87-104 (2007).</li>
<li>Maresca, A., la Morgia, C., Caporali, L., Valentino, M. L., Carelli, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+optic+nerve%3A+a+%26%2334%3Bmito-window%26%2334%3B+on+mitochondrial+neurodegeneration%5BTitle%5D)+AND+%22Mol+Cell+Neurosci%22%5BJournal%5D)">The optic nerve: a &#34;mito-window&#34; on mitochondrial neurodegeneration.</a> Mol Cell Neurosci. 55, 62-76 (2013).</li>
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<li>Vincent, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+biogenesis+and+fission+in+axons+in+cell+culture+and+animal+models+of+diabetic+neuropathy%5BTitle%5D)+AND+%22Acta+Neuropathol%22%5BJournal%5D)">Mitochondrial biogenesis and fission in axons in cell culture and animal models of diabetic neuropathy.</a> Acta Neuropathol. 120 (4), 477-489 (2010).</li>
<li>Leinninger, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria+in+DRG+neurons+undergo+hyperglycemic+mediated+injury+through+Bim%2C+Bax+and+the+fission+protein+Drp1%5BTitle%5D)+AND+%22Neurobiol+Dis%22%5BJournal%5D)">Mitochondria in DRG neurons undergo hyperglycemic mediated injury through Bim, Bax and the fission protein Drp1.</a> Neurobiol Dis. 23, 11-22 (2006).</li>
<li>Leinninger, G. M., Edwards, J. L., Lipshaw, M. J., Feldman, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanisms+of+disease%3A+mitochondria+as+new+therapeutic+targets+in+diabetic+neuropathy%5BTitle%5D)+AND+%22Nat+Clin+Pract+Neurol%22%5BJournal%5D)">Mechanisms of disease: mitochondria as new therapeutic targets in diabetic neuropathy.</a> Nat Clin Pract Neurol. 2, 620-628 (2006).</li>
<li>Edwards, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diabetes+regulates+mitochondrial+biogenesis+and+fission+in+mouse+neurons%5BTitle%5D)+AND+%22Diabetologia%22%5BJournal%5D)">Diabetes regulates mitochondrial biogenesis and fission in mouse neurons.</a> Diabetologia. 53 (1), 160-169 (2010).</li>
<li>Fernyhough, P., Roy Chowdhury, S. K., Schmidt, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+stress+and+the+pathogenesis+of+diabetic+neuropathy%5BTitle%5D)+AND+%22Expert+Rev+Endocrinol+Metab%22%5BJournal%5D)">Mitochondrial stress and the pathogenesis of diabetic neuropathy.</a> Expert Rev Endocrinol Metab. 5 (1), 39-49 (2010).</li>
<li>Schmidt, R. E., Green, K. G., Snipes, L. L., Feng, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neuritic+dystrophy+and+neuronopathy+in+Akita+%28Ins2%28Akita%29%29+diabetic+mouse+sympathetic+ganglia%5BTitle%5D)+AND+%22Exp+Neurol%22%5BJournal%5D)">Neuritic dystrophy and neuronopathy in Akita (Ins2(Akita)) diabetic mouse sympathetic ganglia.</a> Exp Neurol. 216 (1), 207-218 (2009).</li>
<li>Penna, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+benign+prostatic+hyperplasia+stromal+cells+as+inducers+and+targets+of+chronic+immuno-mediated+inflammation%5BTitle%5D)+AND+%22J+Immunol%22%5BJournal%5D)">Human benign prostatic hyperplasia stromal cells as inducers and targets of chronic immuno-mediated inflammation.</a> J Immunol. 182 (7), 4056-4064 (2009).</li>
<li>Lentz, S. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+DNA+%28mtDNA%29+Biogenesis%3A+Visualization+and+Duel+Incorporation+of+BrdU+and+EdU+Into+Newly+Synthesized+mtDNA+In+Vitro%5BTitle%5D)+AND+%22J+Histochem+Cytochem%22%5BJournal%5D)">Mitochondrial DNA (mtDNA) Biogenesis: Visualization and Duel Incorporation of BrdU and EdU Into Newly Synthesized mtDNA In Vitro.</a> J Histochem Cytochem. 58 (2), 207-218 (2010).</li>
<li>Glas, U., Bahr, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitative+study+of+mitochondria+in+rat+liver.+Dry+mass%2C+wet+mass%2C+volume%2C+and+concentration+of+solids%5BTitle%5D)+AND+%22J+Cell+Biol%22%5BJournal%5D)">Quantitative study of mitochondria in rat liver. Dry mass, wet mass, volume, and concentration of solids.</a> J Cell Biol. 29 (3), 507-523 (1966).</li>
<li>Bertoni-Freddari, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Morphological+plasticity+of+synaptic+mitochondria+during+aging%5BTitle%5D)+AND+%22Brain+Research%22%5BJournal%5D)">Morphological plasticity of synaptic mitochondria during aging.</a> Brain Research. 628 (1-2), 193-200 (1993).</li>
<li>Kaasik, A., Safiulina, D., Zharkovsky, A., Veksler, V. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/16870828">Regulation of mitochondrial matrix volume.</a> Am J Physiol. 292 (1), C157-C163 (2007).</li>
<li>Misgeld, T., Kerschensteiner, M., Bareyre, F. M., Burgess, R. W., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+axonal+transport+of+mitochondria+in+vivo%5BTitle%5D)+AND+%22Nat+Meth%22%5BJournal%5D)">Imaging axonal transport of mitochondria in vivo.</a> Nat Meth. 4 (7), 559-561 (2007).</li>
<li>Park, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+swelling+and+microtubule+depolymerization+are+associated+with+energy+depletion+in+axon+degeneration%5BTitle%5D)+AND+%22Neuroscience%22%5BJournal%5D)">Mitochondrial swelling and microtubule depolymerization are associated with energy depletion in axon degeneration.</a> Neuroscience. 238, 258-269 (2013).</li>
<li>Court, F. A., Coleman, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria+as+a+central+sensor+for+axonal+degenerative+stimuli%5BTitle%5D)+AND+%22Trends+Neurosci%22%5BJournal%5D)">Mitochondria as a central sensor for axonal degenerative stimuli.</a> Trends Neurosci. 35 (6), 364-372 (2012).</li>
<li>Baloh, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondrial+dynamics+and+peripheral+neuropathy%5BTitle%5D)+AND+%22Neuroscientist%22%5BJournal%5D)">Mitochondrial dynamics and peripheral neuropathy.</a> Neuroscientist. 14 (1), 12-18 (2008).</li>
<li>Chowdhury, S. K., Smith, D. R., Fernyhough, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+role+of+aberrant+mitochondrial+bioenergetics+in+diabetic+neuropathy%5BTitle%5D)+AND+%22Neurobiol+Dis%22%5BJournal%5D)">The role of aberrant mitochondrial bioenergetics in diabetic neuropathy.</a> Neurobiol Dis. 51, 56-65 (2013).</li>
<li>Kennedy, W. R., Wendelschafer-Crabb, G., Johnson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitation+of+epidermal+nerves+in+diabetic+neuropathy%5BTitle%5D)+AND+%22Neurology%22%5BJournal%5D)">Quantitation of epidermal nerves in diabetic neuropathy.</a> Neurology. 47, 1042-1048 (1996).</li>
<li>Lauria, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((EFNS+guidelines+on+the+use+of+skin+biopsy+in+the+diagnosis+of+peripheral+neuropathy%5BTitle%5D)+AND+%22Eur+J+Neurol%22%5BJournal%5D)">EFNS guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy.</a> Eur J Neurol. 12 (10), 747-758 (2005).</li>
<li>Lauria, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((European+Federation+of+Neurological+Societies%2FPeripheral+Nerve+Society+Guideline+on+the+use+of+skin+biopsy+in+the+diagnosis+of+small+fiber+neuropathy.+Report+of+a+joint+task+force+of+the+European+Federation+of+Neurological+Societies+and+the+Peripheral+Nerve+Society%5BTitle%5D)+AND+%22Eur+J+Neurol%22%5BJournal%5D)">European Federation of Neurological Societies/Peripheral Nerve Society Guideline on the use of skin biopsy in the diagnosis of small fiber neuropathy. Report of a joint task force of the European Federation of Neurological Societies and the Peripheral Nerve Society.</a> Eur J Neurol. 17 (7), e944-e909 (2010).</li>
<li>Umapathi, T., Tan, W. L., Tan, N. C. K., Chan, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determinants+of+epidermal+nerve+fiber+density+in+normal+individuals%5BTitle%5D)+AND+%22Muscle+Nerve%22%5BJournal%5D)">Determinants of epidermal nerve fiber density in normal individuals.</a> Muscle Nerve. 33 (6), 742-746 (2006).</li>
<li>Lauria, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epidermal+innervation%3A+changes+with+aging%2C+topographic+location%2C+and+in+sensory+neuropathy%5BTitle%5D)+AND+%22J+Neurol+Sci%22%5BJournal%5D)">Epidermal innervation: changes with aging, topographic location, and in sensory neuropathy.</a> J Neurol Sci. 164 (2), 172-178 (1999).</li>
<li>Lauria, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intraepidermal+nerve+fiber+density+at+the+distal+leg%3A+a+worldwide+normative+reference+study%5BTitle%5D)+AND+%22J+Peripher+Nerv+Syst%22%5BJournal%5D)">Intraepidermal nerve fiber density at the distal leg: a worldwide normative reference study.</a> J Peripher Nerv Syst. 15 (3), 202-207 (2010).</li>
<li>Hamid, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Hyperglycemia-+and+neuropathy-induced+changes+in+mitochondria+within+sensory+nerves%5BTitle%5D)+AND+%22Ann+Clin+Transl+Neurol%22%5BJournal%5D)">Hyperglycemia- and neuropathy-induced changes in mitochondria within sensory nerves.</a> Ann Clin Transl Neurol. 1 (10), 799-812 (2014).</li>
</ol>

No conflicts of interest to declare.

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 research44,45. This procedure allows for penetration of the antibodies into the tis...

Mitochondria serve vital cellular functions that include producing cell energy, buffering calcium, and regulating necrotic and apoptotic cell death1,2,3. 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 mitochondria within neurons is important.
Over the last 10 years a lot of information has shown that the trafficking and docking of neuronal mitochondria is highly regulated. Motor proteins are involved in distributing mitochondria to specific cellular compartments throughout the neuron. Trafficking of mitochondria is particularly important because neurons project axons and dendrites far away from the soma. Kinesin motor proteins primarily direct anterograde (away from the soma) trafficking of mitochondria along microtubules while dynein motor proteins direct retrograde (toward the soma) motility7,8,9,10. There are cellular signals such a mitochondrial membrane potential and impulse conduction that influence the presence and direction of mitochondrial trafficking11,12,13.
In addition to transporting mitochondria, there are specialized proteins to localize mitochondria to specific cellular compartments that have high energy demands, such as nodes of Ranvier and synapses8,14,17. In fact, the majority of mitochondria within axons are non-motile9,13,18. Specialized proteins like syntaphilin anchor mitochondria to microtubules along axons while other proteins anchor mitochondria to the actin cytoskeleton19-21. Growth factors and ions such as calcium have been reported to support the cessation of mitochondria movement to localize them to regions where they are needed21,22,23.
Taken together, the trafficking and docking of mitochondria are vital for proper function of neurons. In support of this, disruption in mitochondrial trafficking has been associated with several neurological conditions including Alzheimer&#39;s disease, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, Huntington&#39;s disease, hereditary spastic paraparesis, and optic atrophy15,24,25,26,27. Recent studies have focused on mitochondrial dysfunction and pathology as a potential mechanism for diabetic neuropathy, the sensory loss associated with diabetes28,29,30,31,32,33. The hypothesis is that diabetes alters the distribution of mitochondria within the sensory projections of cutaneous nerve ending. Therefore, a technique was developed to visualize and quantify mitochondria within the intraepidermal nerve fibers (IENFs), the distal tips of dorsal root ganglion sensory afferents. The technique combines fluorescence immunohistochemistry of specific mitochondrial and nerve fiber labels with confocal microscopy z-series acquisition of signals with powerful 3D image analysis software to measure the distribution of nerve-specific mitochondria from human cutaneous punch biopsies to achieve this goal.

<table><tbody><tr><td>2% Zamboni&#039;s Fixative</td><td>Newcomer Supply, Middleton, WI&amp;nbsp;</td><td>1459A</td><td>2% paraformaldehyde, 0.2% saturated picric acid in phosphate buffered saline (PBS), pH 7.4</td></tr><tr><td>10x Phosphate Buffered Saline (PBS)&amp;nbsp;</td><td>Fisher Scientific, Pittsburgh, PA</td><td>BP399-4</td><td>To make up 1x PBS</td></tr><tr><td>Image-iT FX Signal Enhancer</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>I36933</td><td>enhances Alexa Fluor dye signals by reducing nonspecific binding</td></tr><tr><td>Anti-Protein Gene Product 9.5 Antibody (Rabbit Polyclonal)</td><td>Proteintech Group Inc. Rosemont, IL</td><td>14730-1-AP</td><td>abbreviated as PGP9.5, replaces discontinued AbD Serotec (Cat. No. 7863-0504) antibody</td></tr><tr><td>Anti-Pyruvate Dehydrogenase E2/E3bp Antibody (Mouse Monoclonal)</td><td>abcam, Cambridge, MA</td><td>ab110333</td><td>abbreviated as PDH</td></tr><tr><td>Goat anti-mouse Secondary antibody Alexa Fluor 594 conjugate</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>A-11034</td><td>red-fluorescent conjugated secondaryantibody</td></tr><tr><td>Goat anti-rabbit Secondary antibody Alexa Fluor 488 conjugate</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>A-11032</td><td>green-fluorescent conjugated secondaryantibody</td></tr><tr><td>Albumin, from Bovine Serum</td><td>Sigma-Aldrich, St. Louis, MO</td><td>A7906-100</td><td>abbreviated as BSA</td></tr><tr><td>Triton X- 100</td><td>Sigma-Aldrich, St. Louis, MO</td><td>T9284</td><td>abbreviated as TX-100</td></tr><tr><td>0.22 &amp;micro;m Filter</td><td>EMD Millipore, Billerica&lt;br/&gt; MA</td><td>MILLEX GP SLGP 033NS</td><td>0.22 &amp;micro;m Millipore filter</td></tr><tr><td>Parafilm M</td><td>Fisher Scientific, Pittsburgh, PA</td><td>13-374-10</td><td>Curwood Wisconsin LLC Parafilm M (PM-996)</td></tr><tr><td>Non-calibrated Loop</td><td>Fisher Scientific, Pittsburgh, PA</td><td>22-032092</td><td>inoculating Loop by Decon LeLoop (MP 199-25)</td></tr><tr><td>96-well Assay Plate</td><td>Corning Incorporated, Corning, NY</td><td>3603</td><td>96-well flat bottom plate</td></tr><tr><td>Prolong Gold antifade reagent with DAPI</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>P-36931</td><td>DAPI staining of nuclei</td></tr><tr><td>Microscope Cover Glass 50 x 24 mm</td><td>Fisher Scientific, Pittsburgh, PA</td><td>12-544E</td><td>Coverslips</td></tr><tr><td>Superfrost Plus Microscope Slides</td><td>Fisher Scientific, Pittsburgh, PA</td><td>12-550-15</td><td>Microscope Slides</td></tr><tr><td>Leica SP5 Laser Scanning Confocal Microscope</td><td>Leica Microsystems, Buffalo Grove, IL</td><td>SP5</td><td>Confocal Microscope</td></tr><tr><td>Volocity x64 Software&amp;nbsp;</td><td>Perkin Elmer, Waltham , MA</td><td>version 4.4.0</td><td>Volocity software is used for Steps 3.1 and 3.2 in the protocol for image processing</td></tr><tr><td>Imaris x64 3 Dimensional Analysis Software</td><td>Bitplane, Concord, MA</td><td>version 7.7.1</td><td>Imaris software is used for Steps 3.3 through 3.5 in the protocol for image analysis</td></tr><tr><td>Excel</td><td>Microsoft, Redmond, WA</td><td>version Office 2013</td><td>Excel spreadsheet software is used for Step 3.6 in the protocol to summarize morphometric features</td></tr><tr><td>Optimum Cutting Temperature Compound</td><td>Sakura Finetek USA, Inc., Torrance, CA</td><td>4583</td><td>abbreviated as OCT</td></tr><tr><td>Leica Cryostat</td><td>Leica Biosystems, Buffalo Grove, IL</td><td>CM1850</td><td>Cryostat for cutting 50 &amp;micro;m sections</td></tr><tr><td>CellLight Mitochondria-GFP, BacMam 2.0</td><td>ThermoFisher Scientific, Waltham, Massachusetts</td><td>C10600</td><td>Used as a postive control to label mitochondria with a green fluorescent signal</td></tr></tbody></table>

three-dimensional imaging and analysis of mitochondria within human intraepidermal nerve fibers

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

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.

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 ...

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.

The goal of this protocol is to study mitochondria within intraepidermal nerve fibers. Therefore, 3D imaging and analysis techniques were developed to ...

The overall goal of this imaging and analysis technique is to isolate specific information from a complex signal. Specifically, this protocol describes how to visualize and quantify nerve-specific mitochondria with other mitochondria within the epidermis. This method can help answer key questions in the field of neurology such as how does mitochondria within intraepidermal nerve fibers of healthy individuals compare to nerve-specific mitochondria of patients with neurological complications.The main advantage of this technique is that we take a complex signal and extract specific information from it. This protocol helps us to isolate a specific signal such as the signal in these straws from the signals around them. Prepare for fluorescence immunohistochemistry staining of skin biopsies by first labeling a 96-well plate according to this schematic.Then pipette 150 microliters of stock signal enhancer solution to reduce nonspecific binding of secondary antibodies into each well of the top row. Prepare rinse wells by adding 150 microliters of 1x phosphate buffered saline into each well in rows two and three of the 96-well plate. Then add 150 microliters of 5%BSA blocking solution into

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Source: Hamid, H. S., et al. Three-dimensional Imaging and Analysis of Mitochondria within Human Intraepidermal Nerve Fibers. J. Vis. Exp. (2017) This video demonstrates the method for visualizing nerve-specific mitochondria using three-dimensional imaging. It outlines the process of acquiring sequential scans of pre-labeled intraepidermal nerve fibers and generating 3D surface images to isolate and display mitochondria in relation to nerve structures.

Visualization of Mitochondria within Intraepidermal Nerve Fibers

In order to study the changes of nociceptive intraepidermal nerve fibers (IENFs) in painful neuropathies (PN), we developed protocols that could directly examine three-dimensional morphological changes observed in nociceptive IENFs.  Three-dimensional analysis of IENFs has the potential to evaluate the morphological changes of IENF in PN.

Three-dimensional Imaging of Nociceptive Intraepidermal Nerve Fibers in Human Skin Biopsies

The protocol for dynamic longitudinal imaging and selective laser lesion of nerve endings in reporter transgenic mice is...

In Vivo Two-Photon Microscopy of Single Nerve Endings in Skin

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals.  Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.



Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Impaired mitochondrial transport and morphology are involved in various neurodegenerative diseases. The presented protocol uses induced pluripotent stem cell-derived forebrain neurons to assess mitochondrial transport and morphology in hereditary spastic paraplegia. This protocol allows characterization of mitochondrial trafficking along axons and analysis of their morphology, which will facilitate the study of neurodegenerative disease.

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

We describe fluorescence photoactivation methods to analyze the axonal transport of neurofilaments in single myelinated axons of peripheral nerves from transgenic mice that express a photoactivatable neurofilament protein.

Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve

Using transgenic fluorescent mice, detailed protocols are described to assess in vivo axonal transport of signaling endosomes and mitochondria within motor and sensory axons of the intact sciatic nerve in live animals.

Expanding the Toolkit for In Vivo Imaging of Axonal Transport

This protocol describes how to reconstruct mitochondrial cristae to achieve 3D imaging with high accuracy, high resolution, and high throughput.

A Three-Dimensional Technique for the Visualization of Mitochondrial Ultrastructural Changes in Pancreatic Cancer Cells

This study presents an in vivo two-photon imaging approach for visualizing the structures of low-threshold mechanoreceptor (LTMR) axon terminals in the forepaw skin of mice. Using repeated, high-resolution imaging of individual axons, this method provides a new platform for studying the structure and function of the cutaneous sensory circuit during development and in adults.

Intravital Two-Photon Imaging of Touch Sensory Axon Morphology in Mouse Skin

Local injections of a mitochondrial dye label terminal Schwann cell mitochondria in vivo for confocal microscopy. This method utilizes proof of concept demonstrated in skeletal muscle tissue from mice with healthy or diseased muscle to provide high-resolution mitochondrial visualization.

Methods for In situ Quantification of Mitochondrial Morphology in Muscle and Terminal Schwann Cells of Mice

Learn three-dimensional imaging and analysis of mitochondria within human intraepidermal nerve fibers using fluorescence immunohistochemistry. This protocol enables researchers to isolate nerve-specific signals and study mitochondrial function in neurological disease.

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

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