[Histonet] RE: trouble with cryosections

[Melissa Gonzalez]

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