Re: [Histonet] Rodent Processing Schedule?

From:Gayle Callis

No, whole rat brains are larger and would require a longer processing 

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 

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
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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