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.
University of Washington
Department of Pathology
> Date: Wed, 30 Jul 2008 09:49:19 -0600
> From: firstname.lastname@example.org
> To: email@example.com
> 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.
> On 7/30/08, Gayle Callis wrote:
> > If you tell us how YOU do it, then we can help modify your protocol.
> > ----- Original Message ----- From: "subat turdi"
> > To:
> > 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
> >> _______________________________________________
> >> Histonet mailing list
> >> Histonet@lists.utsouthwestern.edu
> >> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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