problem with TUNEL assay - (Oct/19/2006 )
hi
i m having trouble with TUNEL assay.... the positive control is DNAse treated and the kit is from droche... when using 2U of DNase and performing the assay ,only the peripheral cells in the coverslip show nuclear staining, while the cells in the middle of coverslip do not pick up signal.... i usually apply sufficient reaction mix on my coverslips and also ensure that it is spread evenly by covering them with parafilm, i also tried increasing the incubation time for both Tdt and the secondary antobody.. but of no use .. can anybody help me in this regard
thanks
Hi.
I think your problem could be the permeabilization treatment. After fixing cells (with 4% PFA in PBS) we use 0.1% Triton and 0.1% sodium citrate (freshly prepared) and incubate 2 min on ice.
Mmm, maybe this work... after cover cells with parafilm incubate 1 hr 37ºC in dark and MOST IMPORTANTLY wet atmosphere. Perhaps only the peripheric cells are in right contact with the atmosphere humidity.
I hope it helps you.
Regards.
i m having trouble with TUNEL assay.... the positive control is DNAse treated and the kit is from droche... when using 2U of DNase and performing the assay ,only the peripheral cells in the coverslip show nuclear staining, while the cells in the middle of coverslip do not pick up signal.... i usually apply sufficient reaction mix on my coverslips and also ensure that it is spread evenly by covering them with parafilm, i also tried increasing the incubation time for both Tdt and the secondary antobody.. but of no use .. can anybody help me in this regard
thanks
I first incubate in Prot K (15ug/mL diluted in PBS) for 15min, wash, then incubate in Tunel rxn mix. Try incubating it in a horizontal slide holder with a little water on the bottom, in the dark, at 37 degress for 1hr. I don't cover the slides because I found it caused the mix to disperse too much. A long as there is some water in the slide holder the slides shouldn't dry out.