Posted by
Stephen C. Kempf on
URL: http://confocal-microscopy-list.275.s1.nabble.com/fixation-of-flatworms-for-confocal-microscopy-tp722505p722634.html
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocalIf permeabilization of your worms turns out to be a problem, it may
help to do a dehydration-rehydration series. We regularly run our
larvae through the following prior to antibody labeling,
distllled H2O
30% ethanol
50% ethanol
70% ethanol
50% ethanol
30% ethanol
distllled H2O
PBS + 0.1% Triton + 0.1% NaN3
PBS + 0.1% Triton + 0.1% NaN3
For our larvae (300 um), 10 min/solution works well. For larger
pieces of tissue, you may want to hold them longer in each solution.
Solutions may be room temperature or cold
We find that this procedure gives better results than just treatment
with Triton.
Steve
On Aug 13, 2008, at 1:16 PM, Julian Smith III wrote:
> Search the CONFOCAL archive at
>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>
> Our worms are much smaller (1mm x 200µm), but we're successfully
> using 4%PFA in 1x PBS with 10% sucrose and use triton X-100 for
> permeabilization.
> If your worms can be anesthetized, this should work fine.
> Julian
>
> Xinyu Zhao wrote:
>> Search the CONFOCAL archive at
>>
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>>
>> Hi, everybody,
>> We have a user trying to do confocal microscopy on flatworms,
>> ( parasitic blood flukes with a size of 1mm x 1cm, Schistosoma
>> mansoni, ). The goal sounds easy: imaging the nucleus of the
>> worms. And I thought the common procedures for immunotaining
>> tissues would apply, i.e., PFA fixation followed by Triton 100x
>> permeablization before the staining.
>>
>> However, in the handful literatures available, people seemed to
>> always use something else, such as an alcohol-formalin-glacial
>> acetic acid solution.
>>
>> Can anybody explain why ? And has anybody tried just PFA and
>> Triton 100X on these creatures?
>> Your input will be very much appreciated.
>>
>>
>>
>> Xinyu Zhao (Jasmine)
>> Biomedical Imaging Core Lab
>> Department of Pathology and Lab Medicine
>> School of Medicine
>> University of Pennsylvania
>> Tel: 215-898-6730
>>
>>
>
>
> --
> Julian P.S. Smith III
> Director, Winthrop Microscopy Facility
> Dept. of Biology
> Winthrop University
> 520 Cherry Rd.
> Rock Hill, SC 29733
>
> 803-323-2111 x6427 (vox)
> 803-323-3448 (fax)
> 803-524-2347 (cell)