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My embryonic and larval shrimp samples are fixed with paraformaldehyde,
stained with rhodamine-phalloidin for the muscles in buffer containing
detergent, dehydrated to methyl salicylate, and imaged with the confocal.
I want to correlate a 3-D understanding of the muscles with their
attachments on the exoskeleton/endoskeleton.  Would that kind of treatment
still be OK for histological sections, or would the detergent be a problem?

Phil H.

At 11:36 AM 10/18/02 -0500, you wrote:
>Search the CONFOCAL archive at
>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
>I also would be interested in going from cleared to paraffin embedded
>and sectioned samples. Given the pre-clearing treatment with K/NaOH
>or trypsin, I'd think the sections would be horrible.
>Phil
>
>>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
>>
>>Phone: (989) 774-2393
>>Fax: (989) 774-3462
>>Email: [log in to unmask]
>
----------------------------
Assistant Professor
Dept. of Biology
Central Michigan University
Brooks 179
Mt. Pleasant, MI 48859

Phone: (989) 774-2393
Fax: (989) 774-3462
Email: [log in to unmask]