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This is especially visible on the intensity profiles where the signals for PADI6 and MATER between the two nuclei (section iii) are almost absent and much more elevated in the cortex (sections i & v) for the O/N 4°C condition.
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The 11 nm nucleosome-like dots can be recognized in the isolated nuclei sections.
For demonstration of DNA content in nuclei, sections were stained with Feulgen's reaction to demonstrate DNA as magenta color.
For detection of the cell nuclei sections were stained with DAPI (1 20,000; Roth, Germany).
After staining the nuclei, sections were mounted and images were captured using a fluorescence confocal microscope.
To detect nuclei, sections were incubated for 10 minutes with TO-PRO-3 iodide (Invitrogen, Paisley, UK).
Intensity measures were taken across four sections for the anteroventral and interpeduncular nuclei sections (i.e., eight total measures for the anteroventral thalamic nuclei for both hemispheres).
The problem of genetic loss due to nuclei sectioning could be overcome by using thicker sections, but this might result in some samples containing more than a single nucleus.
After developing colour in peroxidase substrate for detection of apoptotic nuclei, sections were incubated with the anti-smooth muscle actin (SMA) antibody clone 1A4 (1 400; DAKO) for 1 h at RT.
To distinguish the total number of nuclei, sections were counter-stained with 1 μg/ml 4', 6-Diamidino-2-phenylindole (DAPI) (Molecular Probes, Eugene, Oregon, USA) in PBS for 1 min. Then the slides were washed 3 times for 5 min each in PBS.
D EM image of isolated nuclei ultrathin section.
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