TUNEL assay cell type - HeLa cells human cervical 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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2 years ago

2 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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Found 5 matching solutions for this experiment

Upstream tips
If stored at –20°C, 20X SSC must be warmed to room temperature to ensure that all salts are in solution prior to 10X dilution with deionized H2O.
After opening the kit, 20X SSC can be stored at room temperature.
Protocol tips
You must separate Coplin jars that have been used with DNase from DNase-free Coplin jars.
Upstream tips
- 2.5 × 10^4 cells per well in 24 well plate were seeded.
Protocol tips
- Cells were stained with anti-TDP-43 (1:150), anti-GFP (1:1000), Alexa 647 anti-rabbit (1:1000) and Alexa 488 anti-chicken (1:1000).

- The enzyme solution (terminal transferase) and label solution were mixed in a volume ratio of 1:9
Protocol tips
Fix cells in methanol for 10 min.

Incubate with 2% proteinase K solution for 15 minutes at room temperature.

Incubate with 50 μL of Labeling Reaction Mix at 37˚C for 1 hour in a humidity chamber.
Downstream tips
Analyze by fluorescence microscopy with a 495 nm filter.
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.
Upstream tips
Labeling Reaction Mix, Strep-Fluorescein Solution and TACS-Nuclease Solution should be made just prior to use.
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