[Histonet] Poor quality frozen sections, feline skin.

[Mehlika Faire]

We fix our tissues first in 4% PFA, wash it 3x 15min each with pbs, then place it directly onto 30% Sucrose to cryoprotect until it sinks -usually over night. I am not sure if the Sucrose helps in the quAlity of the sections, but I have dealt with issues similar to what you are dealing with, mostly with snap freeze or meoh/acetone fixed tissues without cryoprotectant.

Hope this helps.
-Mehlika

> Date: Fri, 7 Oct 2011 14:36:24 +1100
> From: julie.bilkey <@t> gmail.com
> To: pbodig <@t> histoprep.com
> Subject: Re: [Histonet] Poor quality frozen sections, feline skin.
> CC: histonet <@t> lists.utsouthwestern.edu
> 
> Hi Peter,
> 
> We use 20-25% sucrose on our fresh specimens for immunofluorescence (IF). We
> only deal with human skin samples so I will not comment on feline skin. I
> sit the specimen in three changes of sucrose for 10 minutes each. Before
> embedding in the cryostat, I gently blot it on a tissue. My understanding is
> that the sucrose does give support like a matrix, and it has saved dried
> specimens for me in the past.
> 
> I hope this helps.
> 
> Julie Bilkey.
> Sydney Skin Pathology.
> 
> On Fri, Oct 7, 2011 at 7:54 AM, Peter Bodig <pbodig <@t> histoprep.com> wrote:
> 
> > I have some feline skin samples for frozen sections that were sent to us
> > because our client was not able to section them themselves. Unfortunately we
> > are having the same problems trying to make good sections ourselves. The
> > samples seem dry and unsupported when we try to cut them. The tissue breaks
> > into small pieces and falls out or the OCT when we try to cut it. We have
> > tried fixation with 10% NBF for various times up to 24 hrs prior to doing
> > frozen sections to give the tissue better support, which helps, but not
> > enough, and then the tissue falls off the coated slides when we try to stain
> > them. Sectioning at 8µ helps as well (that is as thick as out cryostat will
> > go).
> >
> >
> >
> > Does anyone have suggestions on how to get good quality frozen sections
> > from feline skin?
> >
> >
> >
> > We have come up with some questions as well:
> >
> >
> >
> > 1.      Is there a way to support the tissue by infiltrating it with OCT or
> > other similar media to improve our sections?
> > 2.      I have read about using 30% sucrose solution for cryo protection,
> > does that aide in making good quality sections or just reduce freezing
> > artifact?
> > 3.      Does tissue need to be fixed prior to using cryo protection?
> >
> >
> >
> > Our client will be using the sections for Senescence β-Galactosidase
> > Staining.
> >
> >
> >
> > Thank you for any help you can provide,
> >
> >
> >
> >
> >
> > Péter Bodig
> >
> > Laboratory Coordinator
> >
> > Colorado Histo-Prep
> >
> > E-mail: pbodig <@t> histoprep.com
> >
> >
> >
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