Exploded tissue - Summary

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From:"Hendry, Chris I" <HendryC@mar.dfo-mpo.gc.ca> (by way of histonet)
To:histonet <histonet@magicnet.net>
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I'd like to thank everyone for their replies concerning my dilemma.  I've
accumulated the replies (anonymously) I received in case there is someone
else with a similar problem.  Hope this can perhaps help someone else in the
future.  Again, thanks to everyone.
Original Query:
I am doing Paraffin-embedded sections of larval and juvenile marine fish,
and after staining (H&E), analysis of slides reveals that in several, the
tissues have become "exploded", i.e., interstitial spaces have become highly
exaggerated.  Has anyone had experience with this before?  I haven't seen
this before.  Your input would be greatly appreciated.
It sounds like your fixative is hypotonic. The osmotic pressure of a
hypotonic solution will force fluid into your tissue/cells. You should
check the osmolarity to make sure it is equal to or slightly hypertonic
to the cells.
Chris - I can't be sure why you marine tissue "exploded" but it may be a
problem with the tissue having too much water in them.  Some people working
in marine specimens, particularly larval and young, prefer to use sea water
when making 10% formalin instead of tap or distilled water.  You may want to
try that and see if it makes any difference.
There was a discussion about marine tissue on histonet a few months ago and
they talked about fixatives made with seawater. There is a recipe for such
Humason. I'm not at home so I don't have the book nearby. Sorry I can't be
more help but maybe the marine histologists will answer you.
Is the water in your bath too hot?
Have you tried using gelatin in the water bath?
...I have noticed this quite a lot especially with the muscle fibres, I do
not have a
good answer but I do not provide the fixatives and often wonder if this has
anything to do with it, maybe it is not isotonic when used.
This was discussed recently and fixation is probably the culprit.  You need
to fix the marine animals in sea water/formalin, to retain the
isotonicity of the cells of marine animals.  Humason has comments on this
in her book, and how to do the fixation.  Salts are different (higher NaCl)
plus others, you can even rinse the animals with sea water as a buffer
afterwards before processing and do a short processing schedule.
...the problem may be twofold, in that initially your fixative may not be
Isotonic with respect to the Larva and you may have to incorporate sodium
Chloride up to 0.85% in the fixative or use Ultrafiltered Sea Water as your
solvent, I suggest you try the Sodium Chloride method though to allow for
loss of tonicity in sea water due to its dilution with the water content of
Formalin.  The second possibility, is too high a water bath temperature or
infiltration with wax with residual fat solvent (be it Limonene or Xylene
When I am dealing with tissue that is exploding when placed in the water
bath I put a couple of drops of Telpol in the water. This stops the tissue
exploding but causes it to be a little difficult to release the section
from the pick up slide from the 20% Alc to the 40C water bath. But it
works, then I put  the slide in a 37C oven overnight. the result being no
exploded section.
I have worked on cod, haddock and halibut larvae... what fixative
did you use? We usually used a formaldehyde-glutaraldehyde phosphate
buffered EM fix. Specimens embedded in a plastic such as JB 4 worked
better than paraffin, but the latter were usually OK as long as the
times were not too long, or the paraffin too hot. More recently, I have
been working on Tilapia larvae. Here we are interested in LM only, and
found that Bouin's overnight followed by formalin was the best fixative.

Chris Hendry
Department of Fisheries and Oceans
Biological Station
St. Andrews, NB E0G 2X0 Canada
(506) 529-8854 Phone
(506) 529-5862 Fax
e-mail: hendryc@mar.dfo-mpo.gc.ca
URL: http://www.geocities.com/CapeCanaveral/Hall/9440
To steal ideas from one person is plagiarism; to steal from many is


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