Re: [Histonet] cryoprotection before MMA embedding at =?iso-8859-1?Q?-20=B0C?=

From:Gayle Callis

I have never cryoprotected tissues embedded in (Methylmethacrylate) MMA nor
seen a protocol calling for this step with MMA. If you have a publication
reference on this, it would be nice to see their rationale for doing this.  =20

Sucrose cryoprotection is used to reduce water ice crystal formation in
fixed tissue destined for cryosectioning, aka frozen sections.  In general,
after tissue/cells are fixed, you should start dehydration - I am trying to
fathom why sucrose would be used with MMA protocol, awaiting more comments
on this. 

The reason you put MMA in the cold is to prevent it from polymerizing
suddenly as you go through the final steps infiltration steps.  On
occasion, it can suddenly turn into solid plastic - something you do not
want to happen.  Cold temperatures during dehydration and clearing may help
preserve antigens IF you need to IHC, but you still need to remove MMA
before this kind of staining.  

Most people dehydrate, clear at room temperature, but infiltrate at 4C with
MMA to keep the plastic mixture from polymerizing before tissues/cells are
completely infiltrated with unpolymerized MMA. Because heat is given off
during polymerization of MMA, cold embedding is used to protect antigens on
cells IF immunostaining is a goal and can be done at 4C, refrigerator
temperatures also.    

You may have lost the tissue somewhere in the changes of reagents.  

 At 05:59 AM 5/6/2004 -0400, you wrote:
>Hi everyone
>i use a protocol for MMA embedding at 20°C since a few months.
> and this protocol says to put simples in phosphate buffer 7.4 with 10%
sucrose overnight at 4°C after fixation in paraformaldéhyde 4% for 3 days
and before dehydratation and preimpregnation at 4°C,  emebdding is done at
>Last time i forget the step of cryoprotection with sucrose, and after
cutting and staining my embedding simples, i didn't see any cells, no one
cell !!! i did never see that before !!!!
>Do you think that is because of no cryoprotection ?
>Does a lack cryoprotection induce disparition of cells ? the tissue was
very thin... 
>any help would be very usefull
>Thank you very much
>Myriam Baali
>Natural implant
>Histonet mailing list
Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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