[Histonet] Torn tissues followup

From:"Julien Lambrey de Souza"

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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