fresh frozens

From:Karen Larison <>

Hello Histonetters,

I have a researcher who is attempting to duplicate a journal article 
that uses fresh frozen tissue, and the resulting morphology is poor. 
So I'm trying to advise them, but I've have had no direct experience 
with fresh frozens, so my advice is based on things I've heard on the 
HistoNet.  So I'd like some feedback, both on the techniques used in 
the journal article and on whether my advice is sound.

The authors of this journal article are freezing adult rat brains on 
powdered dry ice, and then cutting 12-14 micron sections, and letting 
them "dry" at room temperature for 30 seconds, and then storing them 
at -20C for up to four weeks.  They then thaw their sections for 30 
seconds before immersing them in a very light fixative, which is then 
irradiated in the microwave for 45 seconds.  They then proceed with 


Is it adequate to freeze on dry ice?  And do people routinely store 
their fresh frozen tissue on slides in the freezer?  Wouldn't the 
tissue tend to dry out, and doen't drying wreak havoc on fresh frozen 

The researcher was freezing quadrants of the brain on dry ice, 
putting them in a plastic container wrapped in parafilm, and storing 
at -80C.  Won't this method of storage cause the tissue to desiccate? 
Shouldn't the tissue be immersed in OCT if they plan to store it 

The researcher was collecting 2 sections per slide.  Won't this cause 
the first section to dry, damaging the tissue?

The researcher was storing the slides in a box on dry ice until they 
could store them in the -80C freezer.  Will the colder temperatures 
also cause the sections to dry, causing tissue damage.

The researcher was then letting the box of slides come to room 
temperature before immersing them in fixative.  Again, won't the 
sections dry, thereby causing poor morphology?

So my primary advice to the researchers was that they were doing a 
number of things to dry the tissue before it was adequately fixed, 
and that this was the source of their poor morphology.  Was this the 
correct advice?

A curmudgeon, but not humorless,

Karen in Oregon

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