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Phil

You should be able to go to paraffin from methyl salicylate via 100%
ethanol (several washes) and xylene (a couple of washes). I have no idea
what happens to the fluorescence though... depending on your fluorophore
you may be able to recover the labelling using an antibody to it after
you have cut your paraffin sections (eg an anti-fluorescein or similar
that you can get from Molecular Probes - assuming the fluorescein itself
survives the procedure!)

Good luck

IAN


Phil Hertzler wrote:
>
> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> 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
> ----------------------------
> Assistant Professor
> Dept. of Biology
> Central Michigan University
> Brooks 179
> Mt. Pleasant, MI 48859
>
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--
Professor Ian Gibbins
Anatomy & Histology
Flinders University of South Australia
GPO Box 2100, Adelaide, SA 5001
Australia

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