[Histonet] still having tissue tears

[Favara, Cynthia (NIH/NIAID)]

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.
c

Cynthia Favara
NIAID/NIH/RML/LPVD
903 South 4th Street
Hamilton, MT 59840
406-363-9317


-----Original Message-----
From: Julien Lambrey de Souza [mailto:jlambrey <@t> hotmail.com]
Sent: Thursday, November 13, 2003 12:22 PM
To: histonet <@t> 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 
having.
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.

Chears,
Julien De Souza
University of Quebec at Rimouski.

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