thaw frozen brain in cryostat - freezing artifact

[crispy]

I used to place my frozen mouse brain in the cryostat for about 30min to balance the temperature before slicing. The chamber temperature is ~-21C.   But many of the samples show freezing artifact.   Do i use the cryostat correctly??? 

My mice brains were perfused with cold saline, then 4%PFA (pH7.2), post-fixed in 4% PFA for 24h, cryoprotected in 10%-20%-30% sucrose, frozen in dry-ice cold isopentane for 1-2min. then store in -80C.

Does anyone know whether the freeze-thaw process cause this freezing artifact ??
You guys are so helpful!

-thanks a lot!

[Cardio]

Letting the sample come to -21C should not have caused the freezing artifact. The tissue won't thaw if it was placed directly from the -80 to the cryostat.

Was the tissue in a OCT cast before freezing it with the dry ice slurry?

Others have more experience with freezing brain tissue but one suggestion would be to use isopentane chilled with liquid nitrogen. A general rule is that freezing the tissue faster leads to less artifacts. Be careful not to crack the sample.

[crispy]

Hi,
Thank you for the suggestion!!
Yes, the tissue was placed directly to the cryostat.  But it was not in OCT before freezing.  Is OCT necessary? 

I just learned from this forum that the tissue should be frozen fast, and one method is placing the isopentane in the bath of dry ice and ABS EtOH.  Won't the liquid nitrogen be too cold for the mouse brain?  I guess the tissue will crack if the bath is too cold.

[Cardio]

I have used liquid nitrogen/isopentane for samples that are embedded in OCT. (Muscle not brain). I pea sized sample will take 15 seconds to freeze. To long (30sec-1min) and the sample my crack but not always.

IF the sample is not in OCT prior to freezing then I am guessing you embedded it with OCT at -21C. Then froze the OCT. Correct.

The sample may thaw a little during that phase due to the room temp OCT. The color may change to a different shade if it thawed. ITs obivious in the heart becuase it goes from a pale red to a dark red but with brain tissue it may be harder to see.

I would suggest embedding the sample in a cast mold (OCT) then freezing it with liquid nitrogen/isopentane. You can practice the method with just OCT to get a feel for how long it takes.

Look at this post from CanuckphD http://www.ihcworld.com/smf/index.php/topic,2947.0.html
Are the artifacts chatter or just a shrunken tissue with holes through it. If its chatter then it may be the cutting temp.

[crispy]

The artifacts are holes throughout the brain slice.

Yes, the sample is not in OCT before freezing.  I only used OCT to mount the brain to the specimen discs within the cryostat.

[nzkat]

Hi, I cut adult rat brains on the cryostat and find that if I get horizontal tears/chatters in the tissue sections on the slides this is normally the specimen temp being too cold, or that the brains needed longer to come up from -80 to -20. I cut the brains with the spec temp at -12 C and the chamber temp at -20 and that the brains need a good 3 hours in their before they are a good temp to cut. The brains were snap frozen in powered dry ice. Hope this helps

Kat