Re: [Histonet] still having tissue tears

From:Gayle Callis

Buy the VIR (Veterinary, Industry and Research) Animal Processing Manual
from National Society for Histotechnology, there are several fish
processing schedules, including fixation., the website will tell how to purchase this manaul 

At 07:21 PM 11/13/2003 +0000, you wrote:
>Hello all,
>A few days ago, I wrote to the histonet community to share a problem I am 
>I am trying to obtain nice sections of fish but when I am cutting, the 
>tissue keeps tearing. I have excluded microtome errors since I have tried 
>different blade angles and am using disposable blades (a new one each time). 
>I also keep my parafin blocks cold on ice.
>I think the problem more likely come from the processing.
>Fish (juveniles of a few centimeters long) were previousely fixed in 
>formalin and then transfered to 70% alcohol (a few months ago).
>I cut a centimeter thick piece transversally through the fish.
>This piece is then decalcified for 30 minutes.
>I have tried two processing protocols.
>The first starts in 95% EtoH (70% being skipped since the fish were stored 
>in it)for 1 hour (x2), then 100% EToH for 1 hour (x2), 3 consecutive Xylen 
>baths for 1 hour each and two parafin baths of an hour each, the second 
>being with vacuum.
>Following recomendations given to me after the first histonet message, a 
>second processing protocol was done: 95% EToH (30 minutes), 100% ETOH (30 
>minutes), two xylen baths for 30 minutes each, another xylen bath for 45 
>minutes, parafin for 1H and parafin again with vacuum for 1H30min.
>Both resulted in tissue tearing.
>Does anybody see what I am doing wrong? Does anybody know of a good fish 
>section processing protocol? I have to get this one wright to eventually 
>process smaller and smaller fish, finally ending with larvae processing.
>After cutting, the H&E coloration is really good. Unfortunatly it hasn't 
>been done on good sections.
>Any help would be greatly appreciated.
>Julien De Souza
>University of Quebec at Rimouski.
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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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