TUNEL assay cell type - Rabbit synovial fibroblasts

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 cells in fixation solution for 1 h at +15 to +25°C.

Add tunel reaction mxture and incubate for 60 min at +37°C in a humidified atmosphere in the dark.

Downstream tips
If Nonspecific labeling is noticed, try formalin or glutaraldehyde; reduce concentration of TdT by diluting it 1:2 up to 1:10 with TUNEL Dilution Buffer.

If high background, reduce concentration of labeling mix
to 50% by diluting with TUNEL Dilution Buffer

If Low labeling is noticed, increase incubation time. or incubate at higher temperature
(e.g., +15 to +25°C).

Analyse using a 520–550 nm or 330–385 nm excitation filter, and a 580~ nm or 420~ nm
Upstream tips
- 1.5×10^5 cells were platted in 6cm culture dish
Protocol tips
- Cells were fixed in 1% paraformaldehyde in PBS for 30 min.

- Dehydrate cells in 70% methanol at 4°C for 30 min.
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