holes in white matter

[auburn11]

when looking at spinal cords from animals that were perfused with 4% paraformaldehyde and then cryosectioned and placed onto gelatin-coated slides, we notice holes in the white matter after immunohisto.  When looking at the sections under the microscope after they have dried, but before beginning staining, no holes seem to be present.  If we then place the slides into PBS and mount then the holes become apparent.   Any suggestions/ideas??   

[excalibur]

If you did not cryoprotect with sucrose before freezing, the holes are from ice crystal formation.

[auburn11]

Thanks for the quick response.  We did cryoprotect in sucrose.  We typically place our cords, which have been only perfused fixed (i.e. no post-fixation done), into 15% sucrose overnight and then 30% for 2-3 days.  One thing we have noticed, and to let you know we are in Denver so I don't know the influence altitude may have, but our cords never sink in the 30%.  But after that we go forward and place into OCT and freeze on dry ice.  The molds we use are ones we make out of aluminum foil.     Again when we cut the cords onto gelatin-coated slides the holes in white matter seem to be absent but appear upon rehydration.      Thanks for the help

[richard03]

Ice crystal forms also when frozen step is not quick, i.e., not within 1 minutes.

15% sucrose should be sufficient.

Another concern is freeze & thaw situation which will definitely result in holes on tissue. Make sure your freezer did not power off during storage.

[auburn11]

Just getting back to what might be a freezing issue, as our OCT blocks don't freeze fast enough, definately not within a minute.  We place fixed/cryopreserved rat spinal cords into aluminum foil fabricated tissue blocks and into OCT and then place the blocks onto dry ice.  Is there better system to get faster freezing?  Are there good molds out there for rat spinal cords?  And why would the white matter holes not seem to be present after sectioning but show up when the sections are rehydrated?    Thanks again...

[elfrimml]

We put all tissue directly into isopentane slushy cooled by liquid nitrogen; it freezes very fast, 15 seconds.  Be careful not to freeze dry.  Then we put into OCT & put that in aluminum foil and throw into the liquid nitrogen.  We have no artifacts and as long as we're careful not to freeze dry cutting goes very well.