TUNEL assay cell type - Mouse endothelial cells

TUNEL assay is the cell death detection method where the biochemical marker of apoptosis is DNA fragmentation. The assay involves the microscopical detection of generated DNA fragments with free 3'-hydroxyl residues. in apoptotic cells using enzyme terminal deoxynucleotidyl transferase (TdT) which adds biotinylated nucleotides at the site of DNA breaks. Major challenges of this method involve proper access of the enzyme which could be hampered by poor permeabilization and/or excessive fixation with cross-linking fixative (common with archival tissue). This issue can be resolved by optimizing the incubation time with Proteinase K or CytoninTM.

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1 year ago

1 year ago by Ercole Udinesi Italy

Are 10 week old samples in formalin still usable?

I have left my tumor samples in formalin for an extended period of time (around 10 weeks). Do you think I will be able to use them for TUNEL assay and get results? Thank you for your help in advance

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Found 2 matching solutions for this experiment

Protocol tips
Treat with Proteinase K Solution for 15-30 minutes or Cytonin for 30 minutes.

Treat with Labeling Reaction Mix and incubate for 60
minutes at 37˚C in a humidity chamber.

Treat with TACS Blue LabelTM (page 5), and incubate for 2-7
minute.
Downstream tips
If no staining in TACS Nuclease, Increase incubation time with Proteinase K (up to 60 minutes) or Cytonin™ (up to overnight at 4 oC).
Protocol tips
Add Proteinase K solution and incubate for 15 minutes at room temperature.

Add Labeling Reaction mix and incubate at 37°C for 1 hour.

Add TACS-Sapphire at room temperature..
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