Sucrose cryoprotected, fixed, frozen tissue for paraffin

From:Gayle Callis

I've received an unusual request from a PI who wants to have HE sections 
made from some mouse tissues. The mice were perfused with paraformaldehyde. 

The tissues were then treated with 3 changes of 30% sucrose buffer, then 
frozen for cryosectioning. He uses this method for studies of the brain. He 
wants to look at the rest of the mouse to see if there are any other 
manifestations of the mutation he's studying. The tissues he has are 
already prepared like the brains (perfused, soaked in sucrose, and frozen). 
We could do HE stained frozens, but I wonder if we can get better fine 
detail if we paraffin embed the tissues. So, my question is, has anyone 
tried paraffin embedding and sectioning fixed frozen tissues such as these, 
and, if so, was it worth the effort? (I expect we'll have to remove the 
sucrose first.)
First:  Once again, just drop the frozen tissues into the same PFA fixative
(at RT to do the thawing) you used for perfusion, a postfreezing,
postfixation so to speak. The sucrose is so soluble it should be removed in
the fixative.  To insure total removal, a couple of changes?? might be a
good idea, then proceed to paraffin processing.  Once again, you may see
some artifact in tissues as a result of freezing. 

Second: Just cut a frozen section, mount on a plus charge slide, let it air
dry for a short time, and proceed to H&E staining. Personally, I would try
frozen sections then H&E to avoid any thawing, then paraffin processing.
The sucrose comes out in the distilled water rinse just before hematoxylin.
 A gentle touch without harsh rinsing or agitation as fixed frozens
sometimes dislike staying on slides.  

Good luck, let us know what success you have. 

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (voice mail)
406 994-4303 (FAX)


<< Previous Message | Next Message >>