[Histonet] Processing of mouse hearts

[JR R]

It sounds like over processing to me.  Mouse tissue is easy to over process.

First check the TEMPERATURE of your paraffin pots--they may be running too hot.

Cutting your heart into thin 2mm doughnuts will make the tissue process faster.  I just cut the heart into ventricular half and atrial half--then process the whole thing.  Works great.

Jerry Ricks
Research Scientist
University of Washington
Department of Pathology



> Date: Wed, 30 Jul 2008 09:49:19 -0600
> From: bturdi <@t> gmail.com
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: Fwd: [Histonet] Processing of mouse hearts
> 
> Folks,  I appreciate your responses very much.
> This is the protocol I use.  Please give me some advice what has gone wrong.
> Animals weighed and anesthetized  w/ ketamine-xylazine combo.Chest cavity
> opened quickly and the heart was exposed. IVC was found and KCl was injected
> iv to arrest the  heart was in diastole.The heart was taken out fast and
> placed in a cold PBS to wash out the excess blood. (Perfusion w/ PFA through
> aorta is optional) .The heart was cut into several ~2 mm thick doughnuts
> with special interest on the ventriculum then put into 4% PFA solution for
> 24h at 4 degrees.Then the tissue was rinsed with running water for
> 30min.Then subjected to graded alcohols.
> 
> 70% 30min
> 
> 95% 20min X2
> 
> 100% 20minX2
> 
> Xylene 20min
> 
> Xylene 20 min
> Infiltrate in paraplast:
> Station 1: 45min
> Station 2 : 45min
> Embedding with paraplast
> 
> My major problem is that a lot of times I get dry blocks. The ribbons i get
> often comes without the tissue and breaks into shards.
> 
> Subat
> 
> 
>  On 7/30/08, Gayle Callis <gayle.callis <@t> bresnan.net> wrote:
> >
> > If you tell us how YOU do it, then we can help modify your protocol.
> > ----- Original Message ----- From: "subat turdi" <bturdi <@t> gmail.com>
> > To: <histonet <@t> lists.utsouthwestern.edu>
> > Sent: Tuesday, July 29, 2008 9:31 PM
> > Subject: [Histonet] Processing of mouse hearts
> >
> >
> >  Dear all,
> >>
> >> Does anyone have a detailed protocol for processing mouse hearts for
> >> paraffin slides? I am new to this technique and having various troubles in
> >> getting good histology. We don't have a automatic processer and all is
> >> manual. Thanks in advance.
> >>
> >> Subat  Turdi
> >>
> >> School of Pharmacy
> >> University of Wyoming
> >> Laramie, Wyoming
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> >> Histonet <@t> lists.utsouthwestern.edu
> >> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
> >>
> >
> >
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