Fwd: [Histonet] Processing of mouse hearts

[subat turdi]

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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>
>