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 the wells of row four.
Dilute the primary antibodies PGP9.5 and PDH in 1, 500 microliters of 1%BSA rinsing solution and add 150 microliters to each well in row five. Use an inoculating loop to transfer the sections into the signal enhancer solution in row one. Once the sections are in the primary antibody solution in row five, wrap the plate tightly with laboratory film to prevent evaporation and then agitate the samples on a flat rocker at room temperature for one hour.
Incubate with rocking overnight at four degrees Celsius. Begin day two of the procedure by pipetting 150 microliters of 1%BSA rinsing solution to the wells in rows six, seven, and eight of the 96-well plate. Add the fluorophore-conjugated secondary antibodies to 1, 500 microliters of 1%BSA.
Then pipette 150 microliters of the secondary antibody solution into each well of row nine. Rinse the sections by passing through wells six, seven, and eight. Once the sections are in the secondary antibody solution in row nine, wrap the plate in parafilm and cover with aluminum foil to protect the fluorescence signals.
Incubate with rocking as before. On day three, pipette 150 microliters of sterile filtered 1x PBS into rows 10, 11, and 12. Transfer the samples to row 10 then cover the plate with aluminum foil.
Rinse for one hour at room temperature with rocking. Next, prepare a microscope slide for mounting the sections by pipetting 50 microliters of filtered 1x PBS onto the slide. Once the rinses are complete, transfer a section from row 12 onto the slide then add one to two drops of mounting reagent containing DAPI directly on top of the section.
Gently place a 50 millimeter by 24 millimeter 1.5 microscope glass coverslip over the section. Place the slides in the dark overnight at room temperature to cure the mounting medium before storing or imaging the slides. Select the 40X oil immersion objective on an inverted laser scanning confocal microscope.
Select the appropriate lasers and detectors to image the nuclei, nerve fibers, and mitochondria. Enter the following scan parameters into the microscope software, 12-bit intensity resolution, scan rate of 500 Hertz with two frame averaging, and zoom of 2.2. Set the microscope software for optimized lateral resolution by selecting a scan resolution of 1024 by 1024.
Optimize axial resolution and optical sectioning by selecting a confocal aperture of one air unit with a Z step size of 210 nanometers. Scan each signal separately and adjust the detector voltage and offset to remove any over and under saturated pixels. Activate a live scan for the nerve signal 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.
Scan the final Z series with sequential scanning to eliminate fluorescence signal crosstalk. Isolate the epidermis from the stratum corneum and the dermis by drawing a region around the epidermis using a selection tool on a duplicate image of the original image. Then crop the image to the selection.
Calculate point spread functions for the green and red fluorescence confocal signals using the calculate spread function feature. Then set the parameters for medium refractive index for oil at 1.515 and the numerical aperture for 40X oil objective at 1.25. Set the detector pinhole at one air unit and choose a laser excitation wavelength.
Use iterative restoration set to 100%confidence, iteration limit of 10 cycles, and the green and red PSFs for the deconvulation of the green and red fluorescence signals. Use the create surface tool to make a surface around the deconvolved nerve signals selecting uncheck smooth feature, absolute intensity for thresholding, a lower threshold of 3, 000 and an upper threshold of 65, 535. Keep the surfaces above 10 voxels.
Remove the nonnerve surfaces by using the edit tab in nerve surface to select single nonnerve surfaces or hold down the Control key to select multiple nonnerve surfaces. Delete the selected nonnerve surfaces by pressing the Delete button. Use the edit tab in nerve surface and press the mask all button in the mask properties.
In the new window, select channel five mitochondria deconvolved under the channel selection and then check duplicate channel before applying the mask. Press the radio button for constant inside/outside and check the set voxels outside surface to 0.0 and press the OK button. Finally, create mitochondria-specific surfaces using the create surface tool to make a surface around the masked deconvolved mitochondrial signals by selecting uncheck smooth feature and using background subtraction for thresholding.
Set the diameter of the largest sphere to 1.50 micrometers, the lower threshold to 2, 000 and the upper threshold to maximum 65, 535. Be sure to keep the surfaces above 1.0 voxels. This representative 3D confocal microscopy image illustrates the nerve-specific green fluorescence signal.
A 3D surface shown in cyan is created for the nerve signal. Then the nerve-specific mitochondrial signal is isolated from the rest of the epidermal mitochondrial signals by using the nerve surface as a masking tool. The resulting nerve-specific mitochondrial red fluorescence signal is used to create surfaces shown as magenta around the mitochondria within the nerve surface which is shown in cyan.
This allows the mitochondria in the nerve fibers to be seen in detail. Here, mitochondrial surface data are presented as a size frequency histogram to visualize the percentage of mitochondria that are present in each of the various bends according to their volume. While attempting this procedure, it's important to care for the tissue during the staining process to ensure that the tissue is fully submerged in the solutions to provide even and consistent staining throughout the sections.
After watching this video, you should have a good understanding of how to visualize and quantify mitochondria within human intraepidermal nerve fibers. Our detailed protocol was designed to teach other investigators how to stain, image, process, and analyze human skin biopsies with the goal of understanding the mechanisms underlying the pathogenesis of mitochondrial-based neurologic diseases such as neuropathy.
