TUNEL assay cell type - SKOV3, Caov3 human ovarian 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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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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Found 3 matching solutions for this experiment

DeadEnd™ Fluorometric TUNEL System

Promega

Upstream tips	Protocol tips	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.

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.

Manufacturer protocol Publication protocol 1 Relevant paper

In Situ Cell Death Detection Kit, Fluorescein

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
		- When there is a Nonspecific labelling, reduce concentration of TdT. - When high background is present reduce concentration of labelling mix by 50%. When low labelling is noticed, reduce fixation time.

Downstream tips
- When there is a Nonspecific labelling, reduce concentration of TdT. - When high background is present reduce concentration of labelling mix by 50%. When low labelling is noticed, reduce fixation time.

Manufacturer protocol Publication protocol 1 Relevant paper

ApoBrdU Red DNA Fragmentation Kit

Biovision

Upstream tips	Protocol tips	Downstream tips
- The experiments have been performed three independent times and in triplicates.	-Cells were fixed in 1% (w/v) paraformaldehyde in PBS	- Cells can be stored at –20°C for several days before use

Upstream tips
- The experiments have been performed three independent times and in triplicates.
Protocol tips
-Cells were fixed in 1% (w/v) paraformaldehyde in PBS
Downstream tips
- Cells can be stored at –20°C for several days before use

Manufacturer protocol Publication protocol 1 Relevant paper

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