TUNEL assay cell type - 3T3 L1 mouse adipose 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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Protocol tips
Fix cells in n 4% (w/v) paraformaldehyde for 1 h at room temperature

Permeabilize cells with 0.5% (v/v) Triton X-100 in PBS for 1 h at 37 °C.

Add Tunel mixture and incubate for 1 h at 37 °C in dark.
Protocol tips
- Cells in 4% formaldehyde/PBS at a cell density of 1x10^6 /ml

- Negative controls were performed by substituting Tris buffered saline (TBS) for the TdT enzyme.
Downstream tips
- When there is a loss of tissue from slides, decrease
Proteinase K incubation time.
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