― 271 ―4. Immunoelectron Microscopy:Samples on gold disks were frozen in liquid propane at −175°C, then freeze-substituted with 1% tannic acid in ethanol and 2% water at −80°C for 24 hr. After periods at −20°C and 4°C, samples underwent dehydration in ethanol and resin infiltration at 4°C. They were polymerized at 50°C overnight, sectioned at 90 nm, and placed on nickel grids. Grids were incubated with anti-dsDNA antibody (Abcam) overnight, then with a secondary antibody conjugated to 15-nm gold particles. After staining, grids were observed via transmission electron microscope (TEM) at 100 kV. Images were captured using a CCD camera.The approach involved subtracting Hsp60 and histone-H2B staining images from dsDNA staining images. ImageJ software was used to convert images to binary format and identify dsDNA puncta sized 2 to 20 µm² and with circularity from 0.1 to 1.0. The ‘Analyze Particles’ function aided in this process, with verification from original images if needed. Faint histone H2B signals produced ring-like structures in subtracted images, representing cytosolic dsDNA of nuclear origin, excluded from mitochondrial dsDNA count. The size range could be adjusted based on cell type or histological characteristics. Figure 1E illustrates the method for counting ectopic mitochondrial DNA dots seen in 1B–D.Alternatively, we performed the subtraction of images showing Hsp60 staining from those displaying dsDNA staining, followed by the quantification of dsDNA puncta using the ‘Adjust Threshold’ and ‘Analyze Particles’ functions. Subsequently, we tallied the dsDNA puncta lacking the histone-H2B signal through direct observation. This latter approach is particularly applicable when the histone-H2B images demonstrate a low signal-to-noise ratio. The study introduces a novel method for quantifying cytoplasmic mitochondrial DNA, addressing limitations in existing techniques. Previous approaches lacked quantitative accuracy, with immunofluorescence techniques often insufficient. Droplet PCR and recent protocols using specific markers like LMNB1 and COX4 were explored, but challenges in distinguishing DNA from mitochondria persisted. Hsp60 was adopted to overcome this issue, enabling the assessment of ectopic mitochondrial DNA. Cytoplasmic dsDNA, primarily histone marker-positive, poses challenges for triple staining, with histone H2B utilized here. γH2AX may offer superior performance if staining constraints are resolved. While in situ hybridization is suitable for qualitative assessment, it presents challenges for quantitative evaluation due to potential specimen deformation or noise. Conversely, the immunocytochemical method offers minimal background noise, enhancing quantitative analysis.Results and Discussion# Semi-Quantitative Evaluation of Ectopic Mitochondrial DNA (Fig. 1):
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