[Histonet] Torn tissues followup

[Deborah Faichney]

Hello Julien,

Which species of fish are you working with?  
-We routinely work with a variety of fish and find  different techniques are
required to obtain good sections dependent upon the species, size etc.

When fixing the fish are you opening the abdomen & making an incision into
the head to allow the penetration of fixative? 
-Poorly fixed skin /muscle causes 'holes' and 'tearing' in sections.

We cut, after surface decalcification for approx. 30 min-1hr , at 5 microns
and float out on to distilled water in a heated waterbath.  The slide is
then put into an oven set a couple of degrees above the melting point of the
wax for 1hr prior to staining with H&E. 

When you say,  

''But this methods complicates recognition of sections  to the fish
length.......enables us to keep track of how far we are in the fishe's
body''

-If you need to be able to match serial/consecutive sections to the block
containing one fish you could give each whole fish a unique number/code and
also write the section number to the slide that way you shouldn't have any
problem with identification from original blocks 

eg. 	fish 1 could be 1-1, 1-2, 1-3 etc

	fish 2 could be 2-1, 2-2, 2-3 etc 

Just a few thoughts although it is difficult to help out with your problems
without knowing more information.  if you are interested you could e-mail me
directly and I will send you a copy of our methods.

Hopefully this helps you.

Debbie 


Debbie Faichney
Institute of Aquaculture
University of Stirling
Stirling
Scotland
FK9 4LA 



-----Original Message-----
From: Julien Lambrey de Souza [mailto:jlambrey <@t> hotmail.com]
Sent: 10 November 2003 14:04
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Torn tissues followup


Hello to all histonetters,

I am having problems with torn tissues and I have already posted a message 
asking what may be the causes. Thanks for the various replies. Most of you 
aksed me more details about the procedure in order to try and define a 
problem. so here goes:

Small fish (5cm) where fixed in formalin and then preserved in 70% EtOh

Processing is done on an automated processor
2 hours in 95%alcohol
2 hours in 100%
3 hours in xylene
1 hour in wax (paraplus)
1 hour in wax with vacuum.

Embedding is done with paraplus wax on a histocenter II.

Sectioning is done at 7 microns, transversally to body length.

To mount secions on slides, 2 methods are used. The first is a heated 
waterbath to unrinkle the tape. But this methods complicates recognition of 
sections  to the fish length. That's why we prefer to lay the tapes on a 
black cardboard paper, put some water on slides, put the tape on the slide 
and then heat slightly the slide on a slide warmer so the tape expands to 
unrinkle on the buble of water. This technique has worked fine up to now and

enables us to keep track of how far we are in the fishe's body.

Staining is done on an automated stainer
Xylene 3 min (x3)
100% alcohol 2 min
100% 3 min
95% 2 min (x2)
Water 2 min
Hematoxylin 4 min
Water 2 min
Bluing reagent 2 min
Water 3 min
95% 30 sec
Eosine 2 min
95% 1 min (x2)
100% 1 min
100% 2 min
Xylene 3 min (x3)

Coverslips are mounted automatically.

So there it is. I don't think the tearing is due to microtome blade since I 
use a new disposable blade each time I have a doubt.
So, according to the comments we received up to now, the parameters I'm 
going to change are the processing times in alcohol and xylene. I am going 
to lower them but raise the time in parafin. I have to check the parafin 
temperature as I was told an excessively hot wax will not penetrate as well.

Also I'm going to try and reduce slide warming temperature but leave them 
dry longer, since wet sections may slide off during staining.

So, if you have any further suggestion to help me solve my problem, all 
comments are welcome.

Chears,

Julien De souza.

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