A few drops of a strong base are usually added to clear the paraformaldehyde into solution after it is warmed up.  Look up a recipe for this.  You may not be getting a perfusion, the fluid my be flowing into and out of the heart, with the capilaries blocked by powder or bubbles or red blood cells.

Try a high pressure perfusion.  See the following link:

http://www.myneurolab.com/myneurolab/mnl_products_detail.asp?idproduct=471001&catdesc=Histology+Equipment&CatThreeID=674&CatOneID=4&subcatdesc=Sacrifice+Equipment&idsubcategory=21



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 2:18 PM
To: Histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Sucrose cryoprection

Thanks to everybody who has made me on- and off-list useful and interesting hints. Now it seems clear to me that I have a problem with fixation. I'll try to be more careful; for example making prewash (I do not do that in order to simplify the complicated procedure of perfusing so tiny animals) or checking if there is powder of Paraformaldehyde in the botton (Some times a few grains remain in the bottom). I think that in addition to this the postfixation time must have the key role.

By the way I would like to know your experience with nervous tissue in young animals (1-7 days old in mouse, for example). It is not easy to fix properly this postnatal animals. When I perfuse the paraformaldehyde in adult animals I can see the sad limbs movements which indicate that fixation is going well. In postnatal animals I never get this effect. Have you got any idea regarding this difference? Maybe the more water content in postnatal animals or perhaps -as cleverly a co-lister has suggested me- the more immature CNS system in postanatal animals.

Thanks again

Pablo






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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 




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