I am doing in situ hybridization in plant tissues, but I have questions about this technique. What is the function of triethanolamine used in in situ hybridization. How does this reagent interacts with RNA? Can I change triethanolamine by other reagent?
Thank you.
Hi Piyush,
Thanks for your comments. Well I was facing some problems to do my ISH last year. I verified that is necessary to use Triethanolamina and acetic anydre. I know that it's hard to get it, but you must use it! After I used those reagents my experients worked good, and then I got very nice results. Sure that you must have patient and do your sense and antisense probes to compare. But, if you spend some hours in the lab you able to do it. Please let me know any question concerning ISH. I am very interested and help and learn about this usefull tool.
Regards,
João Paulo
Triethanolamine and acetic anhydride are used as an acetylation step, which I think is used to prevent non-specific background. In the past, we used to include this for isotopically-labelled (radioactive) probes, but we stopped doing this when we switched to digoxigenin-labelled probes. Triethanolamine is a problematic chemical to obtain, I stopped using it and the results have not suffered. In our protocol, proteinase K pretreatment is used for permeabilisation.
I would like to thank all researchers that help me. I will try to find triethanolamine, but if it don't happen I'll do my In Situ Hybridization without it.
I'll try to do my experiment and after that I'll give you a feedback.
Thank you all. Your answers are very important for me.
Sincerely,
Dr. João Paulo R. Marques
Thanks Kristin. I am preparing all solutions and the samples. Thank you one more time.
acetylate with ethanolamine solution for 10 mins (to reduce non-specific binding of the negative probe to the positively charged glass slides and tissue)
you may have a look at the chapter we published
Princivalle et al. 2012;
Detection of mRNA encoding receptors by in situ and Northern hybridization. Methods Mol Biol. 897:261-302.
http://www.ncbi.nlm.nih.gov/pubmed/22674170
Dear Alessandra,
Thank you for the imformation.
I will start to do may In Situ Hybridization in few days. I'll give you a feedback about it.
Best regards,
João Paulo
Dear Joao,
I was wondering what results you got for your In-Situ hybridization without adding triethanolamine and acetic anydride? I have triethanolamine but its difficult to get acetic anhydride because of drug safety issues. Can you please share your experience about your In Situ hybridization?
Hi Piyush,
Thanks for your comments. Well I was facing some problems to do my ISH last year. I verified that is necessary to use Triethanolamina and acetic anydre. I know that it's hard to get it, but you must use it! After I used those reagents my experients worked good, and then I got very nice results. Sure that you must have patient and do your sense and antisense probes to compare. But, if you spend some hours in the lab you able to do it. Please let me know any question concerning ISH. I am very interested and help and learn about this usefull tool.
Regards,
João Paulo
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