Re: Not "rapid" freezing!

From:Robert Schoonhoven

I agree with John completely.  Sometime ago I posted some notes
regarding freezing rates for some cryogenic methods......might be in the
archives somewhere.
I was going to re-post them but I've changed computers since then and I
must not have saved that file.  In short, cells are best frozen in
microseconds not minutes.

"J. A. Kiernan" wrote:

> 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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