TUNEL assay cell type - PC-3 human prostate 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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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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Upstream tips
Seed 10,000 cells in 96-well plate.

Fix sample in 4% formaldehyde for 1 h before freezing.
Protocol tips
Fix sample in 4% formaldehyde for 5 min before using sample.

Block samples in 10% donkey serum in 0.1% Triton-X for 30 min at room temperature.

Incubate primary antibody overnight at 4 °C and with secondary antibodies for 1 h at room temperature
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..
Upstream tips
Seed 1 x 10^5 cells/well
Protocol tips
Treat cells with simvastatin, 10nM docetaxel or a combination of both for 24h.

Fix cells in 2% PFA at 4°C for 30 min
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