Hi everybody,
I am scanning my fish blood cell to calculate the maturity of different cells. This is my first try and I have trouble because various results images with DAPi + WGA cocktail. Hope some can help.
The protocol is after re-hydration blood smear in methanol+PBS, then use triton 1x for 15 min , after that washing and using cocktail DAPi+ WGA to dye the cell.
DAPi give the clear blue color, but WGA (Alexa Fluor 594) can not dye the cytoplasm. As you can see in the photo, The test slide is very good but the second one is not. The red color background I guess is because I did not wash it enough. But why can't WGA dye cytoplasm any more?
Thank for your discussion ideas ^_^!
Hi, I think you have only met with a conventional problem associated with the use of water-soluble fluorophores functionalized with multiple negative charges at physiological pH (2 sulfonates and 1 carboxylate on AF594) which prevents cell penetration due to their surface also negatively charged. Remains in the use of positively charged (or neutral) fluorophores for your protocol.
Good luck
Uhm, I think the problem come at the step DAPi+WGA, but we still don't know why on the first time it is good but next time is so bad. There is not much protocol about fish blood cells available that we can research. :)
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