Whole-mount expansion microscopy

We have always used whole-mount (and confocal) immunomicroscopy in Xenopus, but I am hoping to include expansions (which works great in Xenopus). But imaging is trickier, since the sample becomes opaque once out of clearing agent (salicylate or benzyl alcohol:benzy benzoate.

Anyone have a working method for imaging larger (expanded) samples?

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Specimens naturally become highly transparent during expansion, due both to loss of scattering material and physical dilution. This makes them very well-suited to water-immersion objectives. We normally expand tissue slices with a maximum thickness of ~300um before expansion (~1.2mm after expansion) because anything larger becomes hard to image due to working distance limitations. If you are using a very long working distance objective and do want to expand an entire 1mm-thick specimen, you might need to extend the monomer equilibration time to make sure the monomer solution uniformly permeates the specimen before the onset of gelation. This might in turn require a higher amount of radical inhibitor or a different radical initiator with higher activation energy.