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In situ hybridization - staining (Fast Red) - Which subforum should we post ISH questions anyway??? (Nov/28/2007 )

What is the optimum way to dissolve those Fast Red tablets? I vortexed for a good length of time. Then spun it down at about 1000rpm for a few minutes and got a huge pellet and it started to clear up the solution. I am afraid I'm not getting enough dissolved. Right now I am just using the solution with precipitates as I don't think they will hurt the embryo and I do see some staining. I am using 100mM Tris HCl 8.0~8.2 and have checked the pH. I would spin it or filter it but I'm afraid of losing too much. Is there an optimum pH or solution required to dissolve it? Heating?

Additionally, how do you minimize the horrible orange background in the entire embryos?

Thanks for any ideas.




No, this is for RNA ISH. I'm going to try and see if I can find a company that makes another red producing AP substrate. I've been using the Roche ones. Additionally, I'm going to try and reverse the order of my double in situ. I'll try the BM purple stain first, then do the FastRED one. Maybe the red dissolves over time or just the blue overpowers the red?! Also, I'll try and see if there are other colors or if I could combine fluorescent RNA ISH with chromogenic ones or even double fluorescence.