[Histonet] RE: trouble with cryosections

[Charles Scouten]

Tissue must be snap frozen (within a second or two) to avoid crystal ice
formation in the cells.  Ice in vitreous form is amorphous, and does not
expand on freezing.  Expansion on freezing is a unique property of ice
crystals.  However, vitreous ice is an unstable state of nature.  Even
stored below -80, it will gradually form as crystals, and expand.  The
expansion tears cell membranes, leaks contents, and generally messes up
the tissue for histology, probably also including the cutting
properties.  See the link below for a full discussion

http://www.myneurolab.com/global/Manuals/Tips%20and%20Techniques%20Freez
ing%20Artifact.pdf

Absolutely, cut as soon as possible after freezing as possible, within
hours, not days.

Cordially,
Charles W.  Scouten, Ph.D. 
myNeuroLab.com 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300 x 342
FAX  314 522 0377 
cwscouten <@t> myneurolab.com 
http://www.myneurolab.com 
 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Melissa
Gonzalez
Sent: Thursday, September 29, 2005 12:40 PM
To: histonet <@t> lists.utsouthwestern.edu
Cc: joannew <@t> bluebottle.com
Subject: [Histonet] RE: trouble with cryosections

Dear Joan,
I feel your pain. Today I am also beating my head over mouse skin
sections, which have been fixed in 4% paraformaldehyde and infiltrated
with sucrose. The way I freeze them has varied, as I find discrepancies
which I haven't been able to solve.
 
For one, I find that regardless of how I freeze the samples, if I cut
these difficult tissues right away, they cut beautifully. 
However, If I have to store them in -80, regardless how long they
equilibrate to -20 (we are talking hours up to 2 days) I have trouble
cutting-- the  tissue layers seem to split apart and the OCT doesn't
seem to stick very well to the tissue (To date I have tried abt 6 brands
of OCT,) regardless of the cryostat temperature. 
I don't understand how -80c storage would affect them so much, when they
have been cryoprotected with sucrose, frozen in OCT in either an
isopentane/dry ice slurry or in the cryostat (-20c), placed in sealed
baggies, and put in boxes in the freezer.

So why does everything (incl. any mouse organ) cut so much nicer when I
remove them from sucrose solution when they are ready, embed in OCT,
freeze using isopentane/dry ice, and then cut them that same day? 
Last week I cut these same skins fresh from freezing and embedding, no
problem, and I stored them in -80. Yesterday I removed them from the
-80, left them in -20 overnight, and today I want to scream and tear out
my hair. 

This is not new to me, I have just silently grumbled all these years
(some days are better than others), but I have about 300 skins to get
through and I don't think I can take it!

Thanks in advance for any replies!!

Melissa


-----Original Message-----

   "empty" cryosections! (Joanne Whitehead)
  
Message: 17
Date: Thu, 29 Sep 2005 07:38:22 -0500
From: Joanne Whitehead <joannew <@t> bluebottle.com>
Subject: [Histonet] "empty" cryosections!
To: histonet <@t> lists.utsouthwestern.edu
Message-ID: <1127997502.433be03ea13c6 <@t> bluebottle.com>
Content-Type: text/plain; charset=ISO-8859-1

Hi everyone,
I'm relatively new at cryosectioning, and so far have found it to be a
very frustrating experience! Some days it goes very smoothly and I get
plenty of nice sections, but most often I feel like it's a battle of
wills with the cryostat, which ususally wins.

My major problem is getting "empty" sections - the media cuts smoothly
but the tissue itself curls or crumples up, leaving just an open circle
of media. The other difficulty is with sections wrinkling as they come
off the knife, instead of lying flat. Does anyone know why this happens,
or how I can prevent it? 

I'm sectioning mouse intestinal tissue, which has been rolled into a
"Swiss roll", fixed in paraformaldehyde, infused with sucrose, snap
frozen in isopentane over liquid nitrogen and embedded in OTC. I have
tried cutting at different temperatures, from -18C to -30C, with my
samples equilibrating in the machine at least an hour before I start.


I have seen protocols in which the tissue is embedded in OTC before
freezing, and also infusing the tissue with a mixture of sucrose and OTC
before embedding. Does anyone have any preference for these methods?

I would really appreciate any advice!
Thank you!
 
Joanne
Curie Institute, Paris





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