RE: [Histonet] Sucrose cryoprection

From:"Charles Scouten"

It does sound like the tissue is not fixed, although 48 hours in Paraformaldehyde should be sufficient even without perfusion.  Is the solution fully formed?  Did any white powder remain in the bottom?  It seems you are not getting good fixative.

Did you prewash with saline or sucrose, or just perfuse?  Blood can block many capillaries and block a through perfusion. 


Cordially,
Charles W.  Scouten, Ph.D. 
myNeuroLab.com 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300  
FAX  314 522 0377 
cwscouten@myneurolab.com 
http://www.myneurolab.com 


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Pablo Sánchez Quinteiro
Sent: Thursday, September 30, 2004 4:43 AM
To: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Sucrose cryoprection

Thank for your help Gayle,

I am sorry my mail was not detailed enough.

Yes, I am doing frozen sections with a freezing microtome. The animals are perfused with Paraformaldehyde 4% and postfixed for 48 hours in the same fixative. Then they are transfered to Phosphate buffer.

I have problems when cutting the tissue. The sections (60 microns thick) are apparently nice, but just after putting them into the buffer they do not stay flat and they fall to pieces after gentle touching with the paintbrush.

I have seen that after sinking in sucrose the samples show a gelatin-like appearance. They seem swollen and they are transparent. Could be this related to a poor fixation?

For immunohistochemistry it is not recommended posfixation times greater tan 24 hours, but maybe postnatal or fetal material need longer postfixation time.

Thanks for your interest.

Pablo

>We need more details on exactly what you are doing??????  and what the 
>problems are?  Are you using a cryostat?
>
>At 11:14 AM 9/29/2004, you wrote:
>>Dear listers,
>>
>>I am cutting at the sliding microtome fetal and early postantal mice 
>>brains. Could somebody tell me if is it normal that after sucrose 
>>cryoprection the pieces of tissue show a gelatin-like appearance? I 
>>have lot of problems cutting this tissue.
>>
>>Thanks in advance
>>
>>Pablo
>>
>>
>>
>>
>>_______________________________________________
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>>Histonet@lists.utsouthwestern.edu
>>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>Gayle Callis
>MT,HT,HTL(ASCP)
>Research Histopathology Supervisor
>Veterinary Molecular Biology
>Montana State University - Bozeman
>PO Box 173610
>Bozeman MT 59717-3610
>406 994-6367 (lab with voice mail)
>406 994-4303 (FAX)
>
>
>


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