TUNEL assay from scratch - (Jul/04/2006 )

Hey guys,

Our lab is finally venturing into DNA damage studies and I would really like to do the TUNEL assay. Almost all our neighboring labs are using kits and it’s kinda hard to wheedle something which costs ~$ 600 Cdn for 50 samples. I already have the protocols (several variations of them) for my application (microscopy and FCM) but since this is the first time we’re gonna try it I don’t know if it will be worth doing the assay from scratch. We don’t do a lot of DNA work in our lab and since we have very limited funds, I can’t do any trial and error with kits as expensive as this. I have heard from people that this test is very tricky and tedious and kits are the only way to go. Can anybody share their experience? Thanks a lot.

Micasa

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Hi - here's a cut and paste from my thesis on my TUNEL experiments. They worked pretty well...

Cells were grown on 22 x 22 mm cover slips in 6-well tissue culture plates and treated as described above. Staurosporine was dissolved to 1 mM in DMSO, and diluted 1:2000 in DMEM media supplemented with 0.5% FBS as a positive control for apoptosis (Cotter et al., 1992). Cover slips were washed twice in PBS and cells were fixed in 4% paraformaldehyde in PBS, then rinsed in PBS twice. Cells were then washed in 2X SSC at 80°C for 20 min. Cells were washed with distilled H2O for 10 min then incubated in pre-warmed 0.1 M Tris/0.05 M EDTA at 42°C for 2 min, and rinsed in H2O. Slides were blocked with 1% BSA for 5 min at room temperature, and permeabilised for 2 min at 4°C in 0.1% sodium citrate + 1:1000 Triton X-100. Cells were washed twice in PBS and cover slips were circled once with a PAP pen. Slides were over laid with 50 l of the reaction mix (25 mM CoCl2, 1 mM dATP, 1:100 1 mM FITC-conjugated dUTP, and 12.5 units TdT enzyme (Roche) and incubated for 1 h at 37°C in a humid chamber. Slides were counterstained with 1 g/ml DAPI (in 50% glycerol in PBS) for 10 min, rinsed in PBS, and mounted in mowiol, and viewed using a Zeiss Axioplan 2 fluorescence microscope with selective filters for FITC and DAPI fluorescence.
2.4.16.2 TUNEL Analysis on Histological Sections
Paraffin-embedded sections were sectioned to 5 m and dried onto glass slides. Sections were deparaffinised as previously described (2.4.13.4.2), then incubated in 2X SSC at 80°C for 20 min, after which the protocol described above was followed.

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