FW: [Histonet] fixing sponge larvae for in situs

Ashley Haines ahaines <@t> vims.edu
Fri Jun 9 13:53:54 CDT 2006

Here are some thoughts that I am passing on from a friend with experience in
this area:

-----Original Message-----
From: David Gauthier [mailto:gauthier <@t> vims.edu] 
Sent: Friday, June 09, 2006 2:51 PM
To: 'Ashley Haines'
Subject: RE: [Histonet] fixing sponge larvae for in situs

It sounds like a classical osmolality problem.  The fact that they
blow up in seawater is a little weird, but I'd suggest checking the
osmolality of the fixative vs. whatever the larvae come out of.  It may be
that the 4%PFA is enough to change the osmolality sufficiently and the
larvae are just that fragile.  For something that small, I would think that
1-2% PFA would be sufficient.  You might also ask if their PFA is fresh.  It
can do strange things when it gets old.  Bouins and osmium are both really
bad ideas.  


-----Original Message-----
From: Ashley Haines [mailto:ahaines <@t> vims.edu] 
Sent: Friday, June 09, 2006 2:43 PM
To: 'David Gauthier'
Subject: FW: [Histonet] fixing sponge larvae for in situs

Any thoughts or experience here?

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gary Radice
Sent: Friday, June 09, 2006 2:27 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] fixing sponge larvae for in situs

I have a colleague who wants to do in situ hybridizations on sponge  
larvae  (yes, sponges are animals and they have larval forms!). She  
tried fixing some in 4% paraformaldehyde in sea water, and the larvae  
exploded. She also tried Carnoy's fix, and 4% PFA in Millonig's  
phosphate buffer, and the same thing happened.

The only thing I could think of to suggest was dropping to 1 or 2%  
PFA in sea water. I've seen references to using Bouins for light  
microscopy or osmium tetroxide for TEM, but I don't think either of  
these approaches would be good for in situs.

Any other ideas?

Gary P. Radice			gradice <@t> richmond.edu
Department of Biology	804-289-8107 (voice)
University of Richmond	804-289-8233 (FAX)
Richmond VA 23173	http://www.richmond.edu/~gradice

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