Another thing that can be tried is to gently blot the tissue between paper
towels.  The tissue will stick to the toweling so it will have to be handled
gently when removing it.  We did this routinely.

Rande Kline
EM Science




Phyllis Davie  on 05/14/2002 08:21:48 PM

To:   "Johnson, Teri" , Histonet
      
cc:    (bcc: Rande Kline/EMI/Merck)
Subject:  Re: Specimens in PBS



Teri,
    Fresh tissue left free-floating in saline or whatever, will absorb
excess fluids--sometimes a LOT of excess fluid.  This will cause the tissue
to swell and become distorted, and all that excess fluid may also cause the
freezing artifact you are noticing.  The freezing artifact may be magnified
by less-than-ideal freezing conditions too.  As you mention, the best way to
transport fresh tissues for freezing is on saline-dampened gauze.
    However, in my experience, if you receive a tissue floating in saline,
you MAY be able to recover it by "washing" it in what we call Transport
Buffer.  (I generally have worked with skin and kidney biopsies being sent
for immunofluorescence.  We have people send the specimens in Michele's
media, an ammonium sulfate based media.  The extremely high salt content of
the Michele's media is washed out of the tissues with "Transport Buffer" ).
If you use the transport buffer on the tissues floating in saline, you have
a good chance of reducing or eliminating the ill effects. (wash the tissue
for 3 x 10-minute changes in transport buffer).

I hope this helps.  If you have any questions, or need the recipes let me
know.  I will only send them by attachment because they are so big, so I am
not attaching them to this e-mail since attachments make the histonet
grouchy.

Best of luck,


Phyllis Davie
PhenoPath Laboratories--Seattle, WA
pdavie@phenopath.com



on 5/14/02 1:45 PM, Johnson, Teri at TJJ@Stowers-Institute.org wrote:

> Can someone explain the mechanism involved with having specimens free-floating
> in PBS and why that is not desirable if you will be freezing the specimen
> later?  I've always known that saline or PBS dampened gauze is best for muscle
> biopsies for subsequent frozen section, but I've never known why.
>
> I am dealing with horrible freezing artifact with some specimens today
> (testes), and the only difference between those and previous ones were the
> amount of time spent free-floating in PBS.
>
> Also, is there any reason NOT to cryopreserve fresh, unfixed specimens for
> frozen embedding?
>
> Thanks, as always,
>
> Teri Johnson
> Managing Director Histology Core Facility
> Stowers Institute for Medical Research
> 1000 E. 50th St.
> Kansas City, Missouri  64110
> tjj@stowers-institute.org
>