FW: [Histonet] fixing sponge larvae for in situs

[Ashley Haines]

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.  

Dave

-----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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