TUNEL assay cell type - Islets of langerhans (Beta 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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5 years ago

5 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 sample in 10% buffered formalin for 24h 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
Fix with 4% paraformaldehyde solution or 10% buffered formalin in PBS for 15 minutes at room temperature.

After rTdT reaction mix incubate the slides at 37°C for 60 minutes inside a humidified chamber
Protocol tips
Fix cells in 4% paraformaldehyde and incubated in 0.3% H2O2 in methanol for five minutes to block endogenous peroxidase.

After TdT treatment incubate cells at 37°C incubator for 60 min.
Protocol tips
Apply protinease K for 15 min

Apply TdT enzyme and incubate in a humidified chamber at 37°C for 1 hour.
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