TUNEL assay cell type - HNSCC Detroit 562 human head and neck tumor 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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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
- Cells were pre- treated with various concentrations of rigosertib for 48 hours and stained with TUNEL reaction mixture
Downstream tips
- When Nonspecific labelling is present, reduce concentration of TdT

- When high background is present reduce concentration of labelling mix by 50%
Upstream tips
- Store the Equilibration Buffer, rTdT Enzyme, Biotinylated Nucleotide Mix and Proteinase K at –20°C
Downstream tips
- If high background, Increase blocking time with 0.3% hydrogen peroxide
Protocol tips
Cells were treated with 20 mg/ml proteinase K for 15 min at room temperature after after deparaffinization.

- hydrogen peroxide is applied for 5 min at room temperature.
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