TUNEL problems - (Jul/25/2008 )

Hi,

I am using TUNEL kit from Roche on rat brain and spinal cord fresh frozen tissue. My problem is that I get variable background on the sections within the same staining procedure. i fixate with formaldehyde and permeabilize with ethanol/acetic acid 1/2. Any ideas what the problem could be

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I wish I could help I just wanted to say I have used this kit and have gotten variable results as well. You can try and play around with different fixatives. If you do succeed I would greatly appreciate knowing how you were able to get the good staining as I have been struggling for months with this TUNEL kit and no one seems to know why. (Btw I use cells not tissue, but they say the protocols are very similar.) Best of luck, dont give up (I nearly have).

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Thanks for your reply. Do you know if there is a better kit? I have been looking around at the internet. I have not found any other reports/discussions about this problem?? Can it be that we should mix the TUNEL reaction more with a pipette/centrifuge maybe...

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I recently tried Invitrogen's kit but found the procedure to be a bit more complex. A big problem is that most of these kits are optimized for flow cytometry analysis so if you work with cells or tissue (us) unfortunately it is difficult to find information. You may want to look around for slight changes in fixation protocol. I do not have experience with tissue sections but there is much more information about tissue section fixation than cell cultures so this should be easier for you. I do think the kit is fairly good to be honest it is just the variability in fixation that could be accounting for the background staining. I have noticed the reaction buffer volumes are very very very small. To overcome this problem using the plastic coverslips is useful, i just spread the reagents by applying over the reagents. I am actually going back to using this kit as I had better results with this one than Invitrogen's. Also, do not let anything dry out when doing this protocol, this makes a mess of your results.

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