This is a common problem with mouse tissue. The tissue is being over
processed. There are a few things that you can do to remedy this. One is
to have a dedicated processing schedule for each different tissue that
you process. Although this is ideal, it is often times impossible to
realistically do this. If your lab is processing a variety of different
mouse tissues at the same time, you will frequently encounter over
processed tissue. Although not ideal, a way to get good sections is to
rough into the blocks and before you put them on your cold tray, place
them in your heated water bath for a few seconds. This will rehydrate
the tissue enough so you can get good sections. If you do this often
enough, you will get a "feel" for how long you need to leave them in
your water bath.
It seems like you have a few unnecessary steps in how you prepare these
hearts. Is there a specific reason why you use cold PBS to rinse the
blood out of the heart? Wouldn't formalin rinse the blood off just as
effectively as PBS while also starting the fixation process more
quickly? What is your reasoning for fixing at 4 degrees instead of at
room temp? Lowering the temp slows down the fixation process. The 30
minute running water rinse after fixation is unnecessary as well.
Feel free to contact me if you have additional questions.
Derek
Derek Papalegis HT (ASCP)
Histotechnician
Division of Laboratory Animal Medicine
Tufts University
136 Harrison Avenue
Boston, MA 02111
phone: 617 636-2971
fax: 617 636-8354
subat turdi wrote:
> 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 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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