What can we use except D-limonene? It's basically an organic solvent, but the permeability is actually about the waxy layer (~5 nm) surrounding the vitelline membrane. Would acetone be right for this?
You may use a 2min treatment with heptane.(Limbourg and Zalokar [1973].
or, do as DeLotto 2007
Embryos were dechorionized, permeabilized by rinsing in Isopropanol for 3 seconds and Hexane for 5 seconds. Residual heptane was allowed to evaporate off and embryos were immediately immersed in phosphatebuffered saline (PBS). Under these conditions development proceeded normally for at least 4 hours in PBS
Hi Helena,
Actually I was thinking of not using Heptane or Hexane, because I will be doing live imaging and embryo viability is a real concern here, with Heptane or Hexane, dessication is a big problem, I am aware o the DeLotto protocol. Can you please let me know what do you think I use d-limonene as Matthew Rand did, though I have to look for an alternative for d-limonene, because that's not available here.
Sorry Arijit,
I've never used d-limonene and don't know of other protocols.
best.
Hi Arijit and Helena,
I also never used d-limonene. I usually use methanol to permeabilize that is less aggressive than Heptane. If you want to maintain the embryos alive may be a heat shock is enough, but is only an idea, I never do that
Sure,
I usually permeabilize the embryos after fix it but I supposse that without fixing works too. After wash the embryos in PBT (PBS +0.5%Triton) I wash 1/2 PBT 1/2 Methanol one mitute or two, then retire half of the liquid and add more methanol. Finally I wash the embryos 20 minutes in 100% methanol.
I hope that it works for you!
Try using 25% chlorine, that will at least make the vitelline memberane more permeable.
Aabid, thanks for your answer. Are you sure we can use chlorine for live protocols?
A %age well diluted should not harm the embryo. If uve collected much embryos, a try will not harm u... :-)
Arjit, just to be clear about your question: are you trying to remove the chorion (i.e. the external waxy layer) or specifically permeabilize the vitelline membrane itself after removing the chorion? For live imaging, I've generally removed the chorion by either rolling the embryo carefully on double sided-sticky tape with forceps, or treated with 50% bleach for ~ 5 minutes, then kept the embryo under halocarbon oil. That prevented desiccation, but allowed sufficient gaseous exchange to keep the embryo alive.
Hi Shaad, I was talking about specifically permeabilize the vitelline membrane itself after removing the chorion. I wanted to do large scale de-vitellization, that's why do not prefer sticking the embryos on double sided sticky tape, it sometimes damages the embryos. Anyway, thanks for the halocarbon oil suggestion, we shall look into that.
Hi Arijit,
Was any of proposed method working for you? Were you satisfied with the result?
It would be great if you share your experience!
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