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problem with non-specific binding - ISH at electron microscopic level

Started by auxin, Nov 10 2011 04:05 AM

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#1 auxin

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Posted 10 November 2011 - 04:05 AM

Hello everyone,
I have performed ISH using Dig Oligonucleotide Tailing Kit, 2^nd generation http://www.roche-app...6_12_1_7_2.html for labeling my DNA probe (30bp) and Fab-fragment Sheep-anti-Digoxigenin to visualize labelled sequences under electron microscopy. In case of experiments with antisense probe – the tissues are fully labelled (RT-PCR shows signal only in certain parts of the tissue what is correct for this sequences). Negative control (with sense probe) shows also signal (like in case of antisense probe). Additionally (for no earthly reason!) I have signal after RNase treatment of tissues.

Please help me – what I’m doing wrong?

Many thanks!