[Histonet] still having tissue tears

[Julien Lambrey de Souza]

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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