I am trying to measure calcium in Xenopus around stages 13 - 17 (during neurulation) in relation to an aquaporin my lab is studying. I am injecting 1000 pg of GcAMP6s mRNA into one cell at the four-cell stage into albino Xenopus Laevis embryos. The GcAMP6s I am using was made in 2020. I have not been able to view any fluorescence on timelapses. Live staining of injected embryos with nile blue in the past has shown them maturing normally. Has anyone else had an issue with getting GcAMP6s to fluorescence? Any Ideas for possible fixes would be immensely helpful.
I use a Zeiss steREO Discovery.V8 manual microscope and a Hamamatsu Orca-flash4.0LT camera to take timelapses.
Do you mean GcAMP6s mRNA or plasmid when you say "The GcAMP6s I am using was made in 2020"? The mRNA can not be stored for so long time. You may amplify the plasmid and synthesize new mRNA to do injection.
Hi, thank you for the help. You were correct, I was referring to GcAMP6s mRNA. I'll have to synthesize some more from our plasmids.
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