Is it possible to re-slice cleared samples?

Probes & Prep
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Hi there everyone,

I was thinking about re-slicing samples that have undergone clearing through the DISCO method (vDISCO) in order to image them in a confocal system. Ideally, we would like to register the confocal aquired images to the light-sheet acquired images (The resolution of our Light sheet is not that great for fine sub-cellular details of the cells we are trying to image).

My samples are spinal cords that were not embedded in agarose during the clearing process.

I would like to know if someone has tried (successfully or not) this before. I have some questions concerning the full process:

  1. What should be the medium in which you would ideally re-slice the samples?

I am afraid that using water-based medium (like PBS for example) could re-hydrate the samples (I don’t know if this even makes sense), thus decreasing the quality of the clearing. However, slicing in ECI (Ethyl cinnamate) poses the problem of sticking the sample to the vibratome holder since I don’t think any kind of glue would resist ECI immersion.

  1. Would it be possible to embed somehow the sample in Agar/Agarose after the whole clearing process?

This is somehow related to the first question. I fear that embedding in agarose could re-hydrate the sample thus rendering sub-optimal the clearing quality. Does anyone has experience with this?

  1. Would it be possible to create OCT blocks and cut in a cryostat? I think this could be more simple, though my doubt persist about dehydration. Moreover, to which extent cleared samples are cryoprotected in order to do this?

I am sorry about the structure of my question, I know is kind of disorganized, but I haven’t found information concerning this issue.

Any literature, hint or help would be highly appreciated,

Thank you so much,

J

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Hi J,

I don’t know of any way to section solvent cleared tissue post clearing like this as the tissue becomes very hard and brittle after 3DISCO (or similar) processing.

If you are not married to the DISCO protocols, we showed in our 2022 paper that tissue cleared with EZ Clear can be OCT mounted, sectioned and immunolabeled with similar results to 3DISCO.

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I recall having seen papers that re-embedded in paraffin post disco clearing, but can’t find them anymore.
Here they do something similar:

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Hi there @rlokkie and @jasonkirk ,

Well, I am definitely not married to DISCO protocols, but it has been some time setting it up and I have quite a high number of samples already processed this way. Moreover, since I am boosting the fluorescence by vDISCO, there has been some money invested in these protocols

I think I am going to give a try to the method that they show in the paper attached by @rlokkie, let’s see how the rehydration process works.

Thank you so much both of you for your advices,

Cheers,

J

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Sorry If I am becoming late to this discussion.

I will give you my two cents on this. We have previously sliced cleared tissue with iDisco+. We re-hydrate the sample in PBS-gelatin, then put them in sucrose for cryopreservation before embedding them in gelatin and freezing. You can even recheck the primaries you used in the clear sample, just putting fresh secondaries for incubation; there is no need for a new primary antibody.

Regards,

Edwin