[Histonet] processing

[Gayle Callis]

You are not rambling.   Animal tissues, particularly rodent are very lean, 
and if one really wants to see dry animal tissue, tissues from birds of 
prey (hawks), other birds and reptiles can be among the worst. We custom 
process our rodent tissues, brain, small, etc and have shorter schedules 
for mouse brain versus hamster brain, that type of thing just to avoid 
overdehydration.

The 1 hour per station or change even with 3 X 95% and 3 x 100% never seems 
to bother human species tissues much, but with rodent tissue, it removes 
too much of the bound water.  some larger animals with bigger samples in a 
cassette seem to be ok with standard processing schedules but never with 
heat added.

A curious thing observed over a long career in histowork, I saw the 
addition of heat to processing in automated processor a curious 
thing.  When we used the older carousel (Sp?)  models i.e Technicon, heat 
couldn't be added and processing was done at RT.  True, heat may speed up 
the solvent exchange but it also can add to drying of tissues.  I have 
always wondered why this has become a standard method over the years other 
than trying to speed up processing, but always felt alternating vacuum and 
pressure to be the best way to do that along with paying attention to 
length of processing schedules.

What one needs to do is find the correct balance so that the free water in 
the tissue spaces is removed and not the bound water on protein 
molecules.  Overdehydration and adding heat to processing will exacerbate 
the removal of bound water. Consequently, heat is never added to our animal 
tissue processing steps.




  At 02:38 AM 2/1/2006, you wrote:
>Think rodent tissue can process on the 'hard' side. Wonder what makes it
>'hard'? Is it that the bonds between the proteins are 'stronger' or the
>proteins are 'nearer' together? I mean if we could figure out what made
>tissue 'hard' molecularly then we'd know which the culprit in the processing
>was.
>
>Sorry to ramble.
>
>Kemlo Rogerson
>Pathology Manager
>Ext  3311
>DD   01934 647057
>Mob 07749 754194
>
>
>
>
>-----Original Message-----
>From: Steven Coakley [mailto:sjchtascp <@t> yahoo.com]
>Sent: Tuesday, January 31, 2006 6:56 PM
>To: Histonet <@t> lists.utsouthwestern.edu
>Subject: [Histonet] processing
>
>Good afternoon,
>
>   I'm working with mouse/rat muscle and trying to fine tune my processing
>schecdule.
>   My last run of muscle were very dry.  I have a copy of the NSH Animal
>Processing Manual and trying to strike a balance between those listed on
>page 5-6.  I've noticed most of the protocols do not call for heat or P/V.
>The scedule I currently use is as follows: 1-70% (hold), 1-80%, 2-95%,
>3-100%, 3-xylene all for 30 minutes each and at 38C with P/V.  3 paraffins,
>45 minutes each at 55C with P/V.  I'm fairly new to research and still
>trying to make the "mental" switch in handling the different tissue.
>
>   Thanks everyone,
>
>   Steve
>
>
>
>---------------------------------
>Bring words and photos together (easily) with
>  PhotoMail  - it's free and works with Yahoo! Mail.
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://lists.utsouthwestern.edu/mailman/listinfo/histonet

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367
406 994-4303 (FAX)