TUNEL assay cell type - Mouse skeletal muscle 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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Found 3 matching solutions for this experiment

In Situ Cell Death Detection Kit, Fluorescein

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
Fix sample in 4% formaldehyde for 1 h before freezing.	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

Upstream tips
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

Manufacturer protocol Publication protocol 1 Relevant paper

In Situ Cell Death Detection Kit, TMR red

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	Fix cell samples with Fixation solution for 1 h. Incubate in Permeabilisation solution (0.1% Triton X-100 in PBS) for 2 min on ice. Label and incubate for 60 min at +37°C in a humidified atmosphere in the dark	If low labelling is found, reduce fixation time or try 2% buffered paraformaldehyde

Protocol tips
Fix cell samples with Fixation solution for 1 h. Incubate in Permeabilisation solution (0.1% Triton X-100 in PBS) for 2 min on ice. Label and incubate for 60 min at +37°C in a humidified atmosphere in the dark
Downstream tips
If low labelling is found, reduce fixation time or try 2% buffered paraformaldehyde

Manufacturer protocol Publication protocol 1 Relevant paper

DeadEnd™ Colorimetric TUNEL System

Promega

Upstream tips	Protocol tips	Downstream tips
		- The slides were treated with 2× SSC buffer, 0.3% hydrogen peroxide and then incubated with streptavidin HPR reaction buffer.

Downstream tips
- The slides were treated with 2× SSC buffer, 0.3% hydrogen peroxide and then incubated with streptavidin HPR reaction buffer.

Manufacturer protocol Publication protocol 1 Relevant paper

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