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I also recommend clearing with methyl salicylate for immunofluoresence
imaging of embryonic material from 250 um on up.  What I'm wondering from
the list is: has anyone done a correlative study of a whole mounted
immunofluorescence sample for confocal imaging, then embedded that sample
sample in paraffin for making histological sections.  For me it's not so
important that I retain fluorescence in the paraffin sections, but that
would be great if it worked.

What sort of transition regime works well from methyl salicylate to paraffin?

Phil Hertzler

>   Clearing with methyl salicylate:
>
>   Do vascular injection or other such procedure first. Staining may be done
>   first, or at the appropriate stage of dehydration. (IF staining works.)
>   Depigment with H2O2 in water before deH2O if necessary.
>
>   deH2O through EtOH series:
>                            30%
>                            50%
>                            70%
>                            80% (70% & 80% may be replaced with one 75% step)
>                            95%
>                           100% X2
>   use 24œ48 hours for each step, depending on size of animal; adult
>   Pollimyrus isidori & Cottus bairdi used so far went ca. 48 hours each.
>
>   after deH2O, place animal in 100% methyl salicylate; should clear in
>   18œ24 hours (may take longer if a large animal is used).
>
>   if specimen is or turns cloudy, place in 95% EtOH, repeat final steps of
>   deH2O, then try again.
>
>   there will likely be trapped air bubbles in the specimen--these can be
>   removed with a syringe & needle (don't suck out the air bladder[s]).
>
>   small air bladders (all?) may disappear with time, probably because the
>   methyl salicylate diffuses through the bladder wall & fills the cavity.
>
>Phil
----------------------------
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Dept. of Biology
Central Michigan University
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