Re: Not "rapid" freezing!

From:"J. A. Kiernan"

On Thu, 9 May 2002, Douglas A Gregg wrote:
> All I can say is try it before you knock it. 

I have! Slow freezing artifacts were among the
first things I saw in my first (undergrad) research
endeavour in the early 1960s. There was a striking
difference between sections cut with a freezing
microtome (frozen in seconds with CO2) and the
cryostat (frozen in minutes). My elders and betters
explained it to me, and I've not had a problem
since, with my own work.

My younger colleagues, however, still 
make the mistake of generating freezing artifacts,
and they agonize horribly because it is often
extremely valuable material that they have ruined.
A recent example was some transgenic mouse brains
sent from Italy to Canada in dry ice.

They spend hours looking at the worthless sections,
trying to localize the results of immunohistochemical
stainings. They don't bother to do a simple stain
such as H&E or Giemsa to check if the tissue is OK
before doing the expensive stuff. They weep and
gnash their teeth when they finally recognize that
their slides (typically hundreds of them, bearing
carefully mounted serial sections) are no good for 
anything better than the recycled glass bin.

I feel fully qualified to "knock" slow freezing, on 
the strength of my own experiences and those of the 
many researchers who have asked me (as something of
a "local expert" in their opinions) to advise about
recovering some sort of data from worthless material.
> > > ... whole mouse brain ... will freeze completely in 
> > > about 3-5 minutes.
> > 
> > that could not possibly yield acceptable results for 
> > microscopy. Even with conventional cryoprotection an object
> > that takes 3 minutes to freeze will be full of ice crystal
> > holes. Each hole is about the size of a large cell, and is 
John A. Kiernan
Department of Anatomy & Cell Biology
The University of Western Ontario
London,  Canada   N6A 5C1

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