Histonetters,

this is a follow up re our probelms with artefacts in frozen brain
sections.  We are now getting good sections after sectioning with no
artefacts at that time.  However the investigator brought back the sections
a few days later, after storage at ?$ c in refrigerator, now there are
small holes all over, especially in the cortex.  Not bad freeze thaw
artefacts, but diffuse enought to affect sensitive immunostaining for
specific cell types. Brains were fixed in 4% paraformaldehyde, the to PBS
then to 30% sucrose, frozen in OCT boat in Shandon histobath and direct to
cryostat.  The OCT around the sections now appears wrinkled and has "water"
hole artefacts similar to the tissues.  The brains are from mice infected
with virus.  The problem does not appear to be
preparation/embedding/sectioning related. Any suggestions?.  Is the sucrose
step causing osmotic effects related to the section:OCT gel interface,
hence resulting in subsequent "holes".  Any other insights? Is there a
different embedding/support medium we could use v's OCT?  
Bob    
Dr Robert G. Russell
Director
Histotechnology and Comparative Pathology
Forchheimer  734
Phone: (718) 430-3209
Fax:   (718) 430-3243
E-mail:  russell@aecom.yu.edu

mail address:
Ullmann Building, Room 1005,
Albert Einstein College of Medicine
Jack and Pearl Resnick Campus
1300 Morris Park Av., Bronx, NY, 10461.