[Histonet] long-term storage of rat brain at -80

[Neil Fournier]

I thought many of you might able to help me devise a protocol for long-term storage of flash-frozen perfused rat brains.  I am sure what I am doing currently is not appropriate.  Our procedure thus far is to perfuse rats with saline followed by 4% paraformaldehyde. Post-fix for 48 h and then cryoprotect in 30% sucrose until the brain sinks.  The brains are then removed from sucrose and dried with a Kim Wipe, blocked in the coronal plane, and placed in cryomolds containing OCT.  The mold is then frozen in liquid nitrogen cooled isopentane.  The frozen mold is then placed in a empty ziplock bag and placed in the -80 freezer.  Several months after I did this procedure and sectioned the brains, I found significant holes indicative of freezing artifact in the tissue.   Although I have had some problems in the past with my isopentane freezing protocol,  I have a feeling that this issue is probably the result of storage at -80 degree C but I cannot be for certain.  (I do not believe the issue is perfusion based since we have no issues with microtome sectioned or vibratome sectioned tissue for IHC).     

Could anyone share with me their protocol or provide suggestions?  Lastly, I have looked high and low but does anyone know where I can find some long plastic forceps that would be of adequate length for flash freezing?  Our current one was broken accidentally and none of us know where it came from although we are now beginning to suspect that there might be a mystical histofairy who is responsible for the often magically appearing (and sometimes disappearing) histological chemicals and equipment in labs.  

Thanks again,

Neil