TUNEL assay cell type - Mouse liver tissue

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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4 years ago

4 years 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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Upstream tips
Dry overnight at room
temperature or for at least 2 hours at 45 oC on a slide warme
Protocol tips
Incubate 30 minutes at room temperature.

Add Labeling Reaction Mix and
incubate at 37 ˚C for 1 hour in a humidity chamber
Downstream tips
If no staining in TACS Nuclease, Increase incubation time with Proteinase K (up to maximum 60 minutes) or Cytonin (up to overnight at 4 C).
Protocol tips
- Tissue sections were deparaffinized in xylenes, gradually rehydrated in decreasing concentrations of ethanol,
Downstream tips
- When there is a loss of tissue from slides, decrease
Proteinase K incubation time.
Protocol tips
Fix with fixation solution for 20 minutes at 15-25C.

Add Tunel reaction mixture and incubate for 60min at 37C in a humidified chamber in the dark
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