fixation of flatworms for confocal microscopy

[Xinyu Zhao-2]

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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:  [phone]

[Julian Smith III]

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:  [phone]
>
>  

--
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)

[Renato A. Mortara]

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http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Xinyu, we were successful in fixing different Schistosoma mansoni stages
with 3.5% formaldehyde in PBS (see Bahia et al, Parasitology , 133: 321-329
2006).

Good luck !

Renato

Renato A. Mortara
Parasitology Division
UNIFESP - Escola Paulista de Medicina
Rua Botucatu, 862, 6th floor
São Paulo, SP
04023-062
Brazil
Phone: [phone]
Fax:     [phone]
email: [hidden email]
home page: www.ecb.epm.br/~ramortara


-----Mensagem original-----
De: Confocal Microscopy List [mailto:[hidden email]] Em nome
de Julian Smith III
Enviada em: quarta-feira, 13 de agosto de 2008 15:16
Para: [hidden email]
Assunto: Re: fixation of flatworms for confocal microscopy

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:  [phone]
>
>  

--
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)


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06:43

[Stephen C. Kempf]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

If 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:  [phone]
>>
>>
>
>
> --
> 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)

[Pierre Nourdine Bouchelouche]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi everybody,

we have just purchased an SP5 confocal trying to do calcium measurements in smooth muscle cells.
Do anybody have any experience with calcium measuremenst with SP5.

Thank you.
Best regards
Pierre
Smooth Muscle Research Center
Copenhagen
Denmark

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Fra: Confocal Microscopy List [[hidden email]] På vegne af Xinyu Zhao [[hidden email]]
Sendt: 13. august 2008 20:00
Til: [hidden email]
Emne: fixation of flatworms for confocal microscopy

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:  [phone]