Fwd: Re: [Histonet] Rodent Processing Schedule?

[Atoska S. Gentry]

>To: Gayle Callis <gcallis <@t> montana.edu>
>From: "Atoska S. Gentry" <gentras <@t> vetmed.auburn.edu>
>Subject: Re: [Histonet] Rodent Processing Schedule?
>
>
>Hello Gayle, by now you may have seen my forward to this inquiry. However, 
>the brains I have were fixed in PFA ( I'm not sure what percentage, maybe 
>15%), for approximately 3 weeks prior to processing. Also, please what are 
>the immersion times on your alcohol & clearing agents? Thanks, Atoska
>
>At 10:16 AM 7/30/04, you wrote:
>>No, whole rat brains are larger and would require a longer processing 
>>schedule.
>>
>>We have processed whole mouse brains using  automated VIP (Sakura 
>>Finetek) with NO TEMPERATURE added to dehydration stations nor clearing 
>>as this will add to tissue drying.
>>
>>Make sure your mouse brains are totally fixed, perfusion is ideal, 
>>followed by immersion overnight.
>>
>>We process 70,  80, 95 X 3, 100 X 3, Xylene X 1, Clearite 3 X 1 (or you 
>>can use 2 changes of Clearite 3), and 4 changes of paraffin 30 min X 2 
>>changes, 1 hour x 2 changes.  All stations are at 1 hour with exception 
>>of paraffins, just to shorten total time in that for 3 hours.  Use vacuum 
>>and pressure for all stations, and do not exceed 60C with paraffin 
>>infiltration, avoiding excess heat helps.  What you want to avoid is 
>>removing the bound water on proteins (this leads to hard, dry 
>>tissues)  only free water in tissues spaces.  Some labs like to have 
>>first dehydration stations at lower concentrations of alcohol,  50, 70, 
>>80, 95 X 2, 100 X 2 or 3, clearing, etc.  Xylene tends to harden tissue, 
>>but Clearite 3 (Richard Allan) and Propar (ANATECH) are more forgiving, 
>>single aliphatic hydrocarbon xylene substitutes.  These are only 
>>guidelines as your conditions may be slightly different.
>>
>>Recommended is do some processing runs on normal brain just to see what 
>>is optimal for your fixation and processor conditions.  We recently had 
>>to customize processing schedules after some test runs for both 
>>Periodiate/lysine/paraformaldehyde perfused hamster brain (coronal 
>>sections) and tongue, with a separate schedule for mouse coronal brain 
>>sections.
>>
>>At 07:48 AM 7/30/2004, you wrote:
>>>Hi Everyone:
>>>
>>>Does anyone have a protocol for  processing whole, formalin fixed mouse 
>>>brains? The mouse brains need to be processed whole.
>>>I am concerned that  over-processing, using a schedule meant for human 
>>>brain tissue, will dry the tissue out excessively and make the serial 
>>>sectioning a nightmare.
>>>Also, would the same schedule apply to whole rat brains, or should the 
>>>times be increased a bit?
>>>Thank you.
>>>
>>>Tim Wheelock
>>>Harvard Brain Tissue Resource Center
>>>McLean Hospital
>>>Belmont MA
>>>
>>>
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>>
>>Gayle Callis
>>MT,HT,HTL(ASCP)
>>Research Histopathology Supervisor
>>Veterinary Molecular Biology
>>Montana State University - Bozeman
>>PO Box 173610
>>Bozeman MT 59717-3610
>>406 994-6367 (lab with voice mail)
>>406 994-4303 (FAX)
>>
>>
>>
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>
>Atoska S. Gentry B.S., HT(ASCP)
>Research Assistant III
>Scott-Ritchey Research Center
>College of Veterinary Medicine
>Auburn University, AL  36849
>Phone# (334)844-5579  Fax# (334)844-5850

Atoska S. Gentry B.S., HT(ASCP)
Research Assistant III
Scott-Ritchey Research Center
College of Veterinary Medicine
Auburn University, AL  36849
Phone# (334)844-5579  Fax# (334)844-5850