Mounting in glycerol

After ETC clearing my brain. I mounted it in 85% glycerol (v/v).
The brain was rotating in an incubator at 37°C for 2d.

After 1d the brain was almost completely clear. Just some spots inside looked opaque.

After 2d the brain became opaque.

I was expecting the opaque spots from day one were uncleared parts where the glycerol should diffuse,
but it was the other way around. The brain became opaque and just the outside parts look clarified.

Somebody had the same issue, or has an explanation?

I cleared my brain for 5 days at 37°C, following the protocol and with a standard chamber.


  • nicolasreniernicolasrenier Posts: 44
    edited July 2013
    I had the same experience with Glycerol : it clears the sample shortly and then some opaque precipitate seems to nucleate from inside the sample and spread to the whole brain overnight. However, when I left the sample for a very long time, it became a little clear again (the periphery at least).

    look at the other threads : glycerol doesn't seem to work at all. focusclear is unfortunately so far the only viable option, until we come up with a suitable alternative!
  • rspencerspence Posts: 7
    just wanted to confirm that i as well cleared some tissue but putting it in 85% glycerol indeed caused it to be come white and opaque
  • FabioFabio Posts: 62
    Yes I got some info from Stanford and they told me this is normal.
    The best option for RI matching is FocusClear
  • Hey,
    I need to know how prepare 85% glycerol ?
    85mL glycerol in 100mL dH2O ?
  • AdamAdam Posts: 31
    I've been using 85ml glycerol and 15ml dH20 which works fine.
  • ClaraClara Posts: 18
    Hi everyone, has anyone tried mounting in glicerol for 1mm depth samples, or just for whole brains? Does glicerol work better in thinner samples? thanks!
  • AdamAdam Posts: 31
    Glycerol works much better for thinner samples. In my experience, it's fine up to about 3-4mm, but can't be used for whole or half brains.
  • ClaraClara Posts: 18
    Thanks Adam!! I'll try it :)
  • aklliuaklliu Posts: 1
    I have experienced this before and my tissue block turned a bit opaque and yellow after 37 degrees glycerol immersion for 2 days. So I tested out the optimal time for the glycerol incubation in order to achieve transparency. For a 3mm thick rat brain block, 2-3 hours immersion of glycerol at room temperature would be sufficient to achieve full transparency. Further immersion led to yellowing of tissue, but transparency is still preserved. But overnight immersion, and especially at 37 degrees, will lead to tissue turning opaque.
  • McMooMcMoo Posts: 1
    I am also testing out the optimal time for the 87% glycerol and comparing with Focus Clear. For a 1.5 mm horizontal GFP mouse passively cleared brain takes overnight incubation in glycerol at 37 degrees rendering complete transparency without tissue turning opaque. Using a single photon confocal, I captured the substantia nigra compacta neurons in GFP mouse brains treated with glycerol and focus clear. Glycerol seems to show better neuronal morphology with less autofluorescence than Focus Clear. Prolong storage in glycerol leads to swelling of vascular structures in tissue.
  • IreneIrene Posts: 9
    Hi, i've recently published an article on a new clearing agent (TDE) suitable with clarity that replaces Focus Clear. It is open access, the link is
    Please ask me if you have any questions about it, it is very easy to do and cheap. Furthermore it is suitable with different type of microscopy.
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