TUNEL assay cell type - SKOV3, Caov3 human ovarian cancer

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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Downstream tips
- When there was a little or poor staining, optimise permeabilization step
by adjusting incubation time with permeabilization agent.

- When there is a loss of tissue from slides, decrease
Proteinase K incubation time.
Downstream tips
- When there is a Nonspecific labelling, reduce concentration of TdT.

- When high background is present reduce concentration of labelling mix by 50%.

When low labelling is noticed, reduce fixation time.
Upstream tips
- The experiments have been performed three independent times and in triplicates.
Protocol tips
-Cells were fixed in 1% (w/v) paraformaldehyde in PBS
Downstream tips
- Cells can be stored at –20°C for several days before use
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