Julien,

I agree  with Laurie, but as an alternative, a tungsten carbide tipped
microtome would overcome this problem too.

Best Regards

Alan Bright

Bright Instrument Co.Ltd.
St Margaret's Way
Huntingdon
Cambridgeshire
PE29 6EU
England

Tel No:+44 (0)1480 454528
Fax No:+44 (0)1480 456031
Email: abright@brightinstruments.com
Web Site: www.brightinstruments.com



-----Original Message-----
From: Laurie Reilly [mailto:Laurie.Reilly@jcu.edu.au] 
Sent: 11 November 2003 05:35
To: Julien Lambrey de Souza; histonet@lists.utsouthwestern.edu
Subject: Re: [Histonet] Torn tissues followup


Julien,
You haven't mentioned anything about decalcifying these fish.

When we are to section fish, they are usually fixed in Davidson's or 
Bouin's, both of which contain acids which will decalcify
the bones in the fish. Maybe your torn tissues are caused by the
calcified 
bones damaging your microtome knife.

          Regards,  Laurie.

At 02:04 PM 11/10/03 +0000, Julien Lambrey de Souza wrote:
>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)
>70% 2 min
>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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