TUNEL assay cell type - PANC-1 human pancriatic 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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Upstream tips
Warm slides to 57C for 5 minutes and treate with xylene and ethanol to deparaffinize
Protocol tips
Treat with Cytonin or Proteinase K and incubate at room temperature

Add Labeling Reaction Mix and incubate at 37 ˚C for 1 hour in a humidity chamber
Downstream tips
If no staining in TACS Nuclease, increase incubation time with Proteinase K (up to maximum 60 minutes) or Cytonin (up to overnight at 4 oC)
Protocol tips
Incubate tissue section for 30 min at +37°C with Proteinase K.

Add Tunel reaction and incubate for 60 min at +37°C in a humidified atmosphere in the
Downstream tips
To analyze use an excitation wavelength in the range of 450–500 nm and detection in the range of 515 – 565 nm (green).
MEBSTAIN Apoptosis TUNEL Kit Direct

MBL international corporation

Protocol tips
Add proteinase K and incubate for 30 min at 37C.

Add TdT buffer and incubate for 5-10 min at RT.
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