[Histonet] REPLY: Tearing of fish tissue

From:Gayle Callis

It could be that the decalcification is not complete.  Trim block or take
an already sectioned block, place it face down in decalcifier (formic acid
or HCl, your commerical decalcifier) for a 5 - 10 minutes, rinse it well
with tap water, cool block. Do not change reorientation of block holder so
you can BE SURE to get the first sections that come off the block.  DO NOT
RETRIM, you must have the surface decalcified tissue section, and that is
only for a few micrometers into that block face. If you block face, e.g.
the tissue looks chalky, opaque, you may have poor decalcification, poor
dehydration OR both.  Go back and look at your processing schedule and
decalcification protocol again.   

Rinsing the acid off the block face will save metal parts on microtome from

Good luck,

At 03:09 PM 11/13/2003 -0500, you wrote:
>When you first face the block how does the tissue look? Do they seem dry? I
>am not clear as to what you mean by tearing. Is it like a large section of
>tissue is missing or is there just a streak across the section?
>It might be more helpful if you could take a picture and post so we could
>see what the problem is.
>Just a thought.
>Cynthia Favara
>903 South 4th Street
>Hamilton, MT 59840
>-----Original Message-----
>From: Julien Lambrey de Souza [mailto:jlambrey@hotmail.com]
>Sent: Thursday, November 13, 2003 12:22 PM
>To: histonet@lists.utsouthwestern.edu
>Subject: [Histonet] still having tissue tears
>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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