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Position: Discussion forums > Bioscience biotechniques discussion forums > Immunohistochemistry In Situ Hybridization forum
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Background ISH in sections
Posted by: affaro (IP Hidden, New member, 2)
Date: July 19, 2006 07:46AM
Hello,
I'm new in this forum and I would really appreciate if anyone could help me with my problem. I'm performing mRNA detection by ISH in sections of paraffin-embedded tissue blocks (zebrafish). I use DIG-labeled probes and detection by AP-conjugated antibody anti-DIG and NBT/BCIP for staining reaction. I’m blocking with 1% Blocking Reagent (roche) diluted in TBST. It seems I have a problem in the staining step. A few hours after added the NBT/BCIP solution the sections start developing a brownish color and if I leave the NBT/BCIP solution overnight (as the original protocol suggests) I end up with some slides completely “burned” being impossible to distinguish properly the cell limits. This only happens in the slides incubated with RNA (sense or antisense probes) but not in negative controls incubated without DIG-labeled probe. In some ISH it is possible to distinguish a blue signal, but I can never get rid of the brownish color in the tissue that appears like a sort of background. If any of you have any clue on how can I solve this problem, please let me know. Many thanks.
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