Re: fresh frozens

From:Lorraine Gibbs <>

Hi Karen-

I do a lot of mouse brain. Yes, we frequently freeze rodent brain by burying
it in powdered dry ice. When I freeze other tissues, I do isopentane and
liquid nitrogen and  with some just liquid nitrogen. I usually stick with
just dry ice for rodent brains (whether fresh or fixed + sucrose) because
brains as well as larger pieces of tissue tend to crack with rapid freezing.
Sometimes, we collect as many as eight sections per slide. The sections stay
at room temp for a half hour or so. The unfixed sections are fine at RT. For
in situ, we minimize time at room temp and post-fix before storing in
Whether the sections are fixed prior to sectioning, or post-fixed within
minutes of sectioning, or air-dried without fixation, they all get stored in
a -70C freezer. I place slides in plastic slide boxes and seal with tape.
When the slides are retrieved from the freezer, I allow condensation to
evaporate before the slides are fixed. OCT: sometimes brains section better
without it. Sometimes we use it for small pieces of brain but I prefer to
section large brains without any surrounding media.

Good Luck!

Lorraine Gibbs
Physiology and Biophysics
University of Washington
----- Original Message -----
From: "Karen Larison" <>
To: <>
Sent: Friday, May 25, 2001 4:23 PM
Subject: fresh frozens

> 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
> IHC.
> Questions:
> 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
> tissue?
> 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
> frozen?
> 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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