Skin prep for confocal

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des
des's picture
Skin prep for confocal

Hi, my name is Des and I am a PhD student from Ireland. I specialise in Drug Delivery, and have very little experience in microscopy. I was hoping to pick your brain with regards to some fluorescent images I would like to obtain? Basically I wish to insert 200um needles into the skin which have been coated with a lipophilic dye eg nile red. Upon removal of the solid needles, the dye will be retained in the skin. I then wish to freeze the skin, and view cryosections so I can see the fluorescent imprint of the needles. My question is, what would be the best way to stain the tissue so that it can still be frozen? I dont need an awful lot of detail if I could see layers of skin, with a needle shaped imprint I would be delighted. Would it be possible to fix the tissue in glut, insert and remove needles, freeze and then cryosection. From what Ive read the tissue should show up green?

Any input would be greatly appreciated.

Jason King
Jason King's picture
We used to see very clear

We used to see very clear needle tracks in mouse tumours that we had injected with lacZ expressing viruses (Ad mostly). In this case the whole tumours were fixed in glutaraldehyde and incubated in lacZ staining solution (containing X-Gal). Nice needle tracks were visible within 2 hours but we left the overnight (i think).

The question that I'm not sure about is whether you should needle the skin first and then fix or the other way round? If you fix first then you may change the ability of the dye to bind lipids (I'm just guessing though). Doing it the other way, one would need to be sure that the fixing agent did not destroy the dye colour.

When I've done cryo on mouse lung tissue, I've coated the lungs with OCT and snap frozen in liquid N2. After cutting 10-15 micron sections I have pipetted on to the tissue slice about 100 L of methanol and then removed it after about 30 seconds - this fixed the sections well and did not damage the GFP fluorescence I was looking for.

Good luck though.