Our laboratory investigates the cellular mechanism involving protein aggregation in the context of Parkinson's disease. In addition, we use cellular and animal models for testing potential treatments against Parkinson's disease. In this article, we propose a protocol for the study of mitochondrial morphology in situ as an indicator of their health based on immunostaining and image analysis in paraffin-embedded tissue sections.
Mitochondrial morphology has been extensively studied in Parkinson's disease and other diseases in vitro. Nevertheless, protocols for studying mitochondrial morphology in vivo are still lacking. This protocol provides a robust method for staining mitochondria in brain tissue, and it explains in details how to perform the morphological analysis.
This study shows that immunostaining combined with image analysis is a reliable method for understanding mitochondrial morphology. In fact, it allows to quantify the number of mitochondria as well as other morphological parameters, such as the aspect ratio in both cell culture and tissue. Importantly, as this method is immunofluorescence based, it is possible to relate the morphologies back to specific cellular populations.
Here we present a protocol for studying mitochondrial morphology in Parkinson's disease, but this protocol can be applied to any in vivo disease model. In addition, it may aid screening lead compounds for the treatment of not only Parkinson disease, but also any other disease characterized by mitochondrial dysregulation. To begin, take the paraffin brain sections of PFF-injected C57 black six mice.
Dewax the slides by dipping them in xylene substitute and incubating them for two minutes. For rehydration, dip the slides in 100%95%and 70%ethanol, and then two times in deionized water. Transfer the samples into a microwaveable container along with double distilled water.
Warm the 100 times citrate buffer with pH six at four degrees Celsius. Then prepare 350 milliliters of fresh citrate buffer. Pour the prepared citrate buffer into a plastic container with a lid.
Place the slides into the citrate buffer container and secure the lid. Microwave the samples at 700 watts for four minutes, and then allow time for a five minute rest. Microwave for another 1.5 minutes, followed by a five minute rest period, and replenish the citrate buffer.
Microwave for 1.5 minutes, and let it rest for five minutes. Then cool the samples and citrate buffer on ice for 20 minutes and wash them with double distilled water. Dry the sample slides using a paper towel without touching the tissue.
Using a pap pen, draw rectangles around the pieces of tissue. Now transfer all slides to a slide immune-staining box. Gently wash them multiple times with TBS-T ensuring TBS-T drops stay within the pap pen rectangles.
Tap the slides on a paper towel to remove TBS-T. Add 50 microliters of blocking to each rectangle and incubate for one hour at room temperature. At the end of the incubation, using a paper towel, remove the blocking solution from the samples.
Gently pipette 50 microliters of the primary antibody mixture in TBS-T to each rectangle and incubate the samples overnight at four degrees Celsius. Then wash the slides with TBS-T. Remove the TBS-T by tapping on a paper towel.
Repeat this process three times. Next, add 50 microliters of secondary antibody mixture at one to 1000 dilution in TBS-T to each rectangle and incubate at 37 degrees Celsius for one hour in the dark. Afterward, wash the sample three times with TBS-T.
Then incubate the sample with DAPI at a concentration of two micrograms per milliliter and TBS-T for one minute. Remove the DAPI solution by tapping on a paper towel and wash three times with TBS-T. To mount the glass cover slip, add two drops per slide of mounting reagent to the samples.
Press the cover slip gently to eliminate bubbles and let the samples dry for one hour at room temperature. Image at least 50 cells in various fields using a structured illumination fluorescence imaging system equipped with a 60 times oil objective or a confocal technology. To analyze the cells, isolate the region of interest by drawing a contour around the positive or negative cell and using the crop function.
Next, activate shape descriptors and limit to threshold options in the set measurement function. Adjust the threshold level by selecting the threshold function in the adjust menu. Finally, use the analyze particles tool and set an appropriate size to capture the mitochondria.
For example, set the size to 25 infinity, then activate pixel units and select show masks. Substantia nigra pars compacta of PFF injected mice co-immunostained for tyrosine Hydroxylase, VDAC1, PS129 alpha-synuclein and DAPI are shown. Anti PS129 alpha-synuclein staining allowed to discriminate cells that harbored PS129 lesions from healthy cells.
Image analysis of VDAC1 staining of tyrosine Hydroxylase positive neurons revealed a reduction of both mitochondrial number counts and aspect ratio between neurons bearing PS129 lesions and neurons lacking them.
