4 In Situ Nick End-Labeling: Electron Microscopical
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7. Incubate in normal sheep serum, for 15 mins.
8. Incubate in the anti-digoxigenin antibody conjugated to colloidal gold diluted to 1 : 50 with Tris-BSA for 60 mins.
9. Rinse in Tris-BSA buffer for 5 mins.
10. Rinse in distilled water, for 5 mins, three times.
11. Stain with uranyl acetate for 5 mins and then stain with lead
citrate for 1 min.
12. Wash with distilled water in a beaker.
Image analysis via NIH image program
1. Observe the ultra-thin sections using a TEM and take photographs of the labeled cell at high magnification (X 15,000 to
20,000X) (Fig. 4).
2. Import the images of the whole cell from the electron micrographs on a computer via a scanner.
3. Leave only the image of the nucleus on a computer using a
soft (Fig. 5).
4. Automatically measure the nuclear area (11m2 ) and count
the number of the immunogold particles via NIH Image program (Fig. 6).
5. Calculate the labeling density for DNA strand breaks (the
number of immunogold particles / 11m2).
Results
In the conventional ISEL technique, the reaction products of
DAB oxidized by horseradish peroxidase demonstrate the localization of 3' -OH DNA ends in apoptotic cells as a brown color
(DAB reaction).
• Melanosomes of epidermal cells and granules of eosinophils
with peroxidase activity show a false positive 3, 3' -diaminobenzidine (DAB) reaction when they are incubated with a solution containing DAB (Fig. 7). Therefore, the conventional