I would like to analyze a 1mm in thickness skin sample using confocal microscopy. The sample is too thick to be placed between a slide and coverslip then sealed by nail polish. The idea is to do z-staking and I don't want to cut the sample. Is there is an alternative way to tackle this issue? For example, can I mount the sample using some sort of adhesive tape?
Thanks
Hi Mays,
Depending on what the sample is you could add small "feet" made out of folded aluminum foil to add space between the coverslip and the slide to provide uniform support to the coverslip. This could be sealed with fingernail polish if its not to hit pr you can seal it with tape on both sides. We customarily do this with pulled plant bark from small stems. Another option, if available, would be to use a confocal that is equipt with an inverted microscope rather than an upright.
1 mm isn't that thick. If you don't need to recover the tissue, mounting it in a hardening mounting medium like prolong will work fine and give you the added benefit of anti-fade (and refractive index matching if you will be using an oil immersion lens).
If your confocal is on an inverted scope you can also place the tissue in a 35 mm glass bottom dish in solution and immobilize it by placing something like these slice anchors on top: https://www.warneronline.com/slice-anchors-kits-harps
I just followed the routine mounting procedure for the rat WHOLE mesenteric branch artery (MBA) and did the z-stacking in confocal scope and obtained good results and published. You might want to check the below articles for the protocol.
Identification and characterization of novel perivascular adventitial cells in the whole mount mesenteric branch artery using immunofluorescent staining and scanning confocal microscopy imaging - PubMed (nih.gov)
IJCB2012-172746.pdf (nih.gov)
Adventitial neuronal somata - PubMed (nih.gov)
You can add ‘spacers’ as mounts to raise up the coverslip. Use a diamond scribe glass cutter to cut slices of coverslip. Mount these on your slide using nail polish or glycerol in parallel so your coverslip will rest on top of them with your sample in between in mounting media. Then seal round the edges with glycerol gelatine.
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