[Histonet] Torn tissues followup

[Lesley Weston]

As well as the bones, perhaps the scales are causing trouble too; they are
also calcified. Either way, decalcification should solve the problem.

Lesley Weston.



on 10/11/2003 9:34 PM, Laurie Reilly at Laurie.Reilly <@t> jcu.edu.au wrote:

> 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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> 
> 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. 
> 4811                                      laurie.reilly <@t> jcu.edu.au
> 
> Australia.
> 
> 
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