Ultrastructural identification of apoptotic nuclei using the TUNEL technique

Area of Science:

• Cell Biology
• Molecular Biology
• Histology

Background:

• DNA fragmentation is a hallmark of apoptosis.
• Existing methods for detecting DNA fragmentation have limitations in sensitivity and early detection.
• Ultrastructural analysis offers high-resolution insights into cellular processes.

Purpose of the Study:

• To develop and validate an ultrastructural adaptation of the TUNEL assay.
• To enable early detection of DNA fragmentation in apoptosis.
• To identify apoptotic cells before classical morphological changes are evident.

Main Methods:

• Adaptation of the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling (TUNEL) technique.
• Embedding tissue in hydrophilic resin for thin sectioning.
• Biotinylated dUTP labeling followed by avidin-gold particle conjugation for ultrastructural visualization.

Main Results:

• The adapted TUNEL method successfully labeled DNA fragmentation in apoptotic cells.
• Specific labeling of heterochromatin near the nuclear envelope and nucleolus was observed.
• The technique identified DNA fragmentation in cells with normal morphology, suggesting early apoptotic events.
• Validation was performed using L929-8 cells treated with tumor necrosis factor alpha and chick embryo tissues.

Conclusions:

• The enhanced ultrastructural TUNEL method allows for early detection of DNA fragmentation.
• This technique is valuable for identifying cells undergoing apoptosis before overt morphological changes.
• It can detect DNA fragmentation in cells that do not exhibit classical apoptotic characteristics.
• The method, when combined with other techniques, aids in the comprehensive study of apoptosis.