Fwd: [Histonet] Processing of mouse hearts

[Monfils, Paul]

> ----------
> From: 	histonet-bounces <@t> lists.utsouthwestern.edu on behalf of Derek Papalegis
> Sent: 	Wednesday, July 30, 2008 12:52 PM
> To: 	subat turdi
> Cc: 	histonet <@t> lists.utsouthwestern.edu
> Subject: 	Re: Fwd: [Histonet] Processing of mouse hearts
> 
> 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 <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
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> >>>
> >>>       > 
> >>     
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