[Histonet] fixing sponge larvae for in situs

[Esther Peters]

Dear Gary,

Is your colleague working on marine or freshwater sponge larvae?

The osmolarity of the fixative, as someone else on the list mentioned,
is key.  Even 1-2% PFA in seawater might adversely affect the larvae.
It would be best to check the osmolarity of the fixative using an
osmometer to get it as close as possible to the seawater they are in (or
freshwater, if that is the case).

I have fixed corals for years and we have some "unknowns" in some of the
sections that we suspect are marine sponge larvae.  These were fixed
using Helly's (potassium dichromate, zinc chloride, and formaldehyde).
They are just 1 mm or less round to ovoid aggregations of cells, not
much CT (spongin) at all, so might be very delicate to handle in a water
sample and might be "exploding" because of the way they are handled
post-fixation (how are they removed from the fixative and processed)?

We are now using Z-Fix concentrate diluted 1 part to 4 parts filtered
seawater for our coral fixations.  This might be okay for the sponge
larvae?

We also use this fixative, as well as others, on coral larvae with
excellent results and have never had to anesthetize them before fixation
(as Rene had suggested).  But the epithelial cells in coral (and other
cnidarian planulae (term for these larvae) are anchored to a layer of
primitive connective tissue, or mesoglea, within 24 hours or so of
fertilization.

Would be interested in learning if you find a successful fixation protocol!

Esther Peters, Ph.D.
George Mason University

Gary Radice wrote:

  > 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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