Re: [Histonet] Torn tissues followup

From:Laurie Reilly

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.
>Julien De souza.
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Mr.Laurie Reilly                                              Ph 07 4781 4468
Physiology & Pharmacology                           Fax  07 4779  1526
Aust.Inst.of Tropical Vet.& Animal Sc.
James Cook University
Townsville  Qld. 


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