[Coral-List] Technical question about fixation of coral tissues for EM

[Eric Borneman]

HI Alina:

I used parformaldehyde/glutaraldehyde to fix and then left in in 0.4M  
sodium cacodyllic acid and 4%NaCl as a wash buffer and then left it  
longest in buffer. For me, the chelation with EDTA took way too long  
to do in the field since I was using coral colony and not larvae -  
but even after decalcification I left the tissues in a 0.4M phosphate  
wash buffer. Once I started with osmium, I fixed for one hour with  
trimmed samples and went immediately through dehydration steps into  
propylene oxide so there was no time to let anything sit after  
postfixation. I embedded with EPON 812 to prevent wrinkling of the  
thin tissue (A. cervicornis, S. pistillata. P. damicornis). I'm sure  
others do it differently or better, but I left parts of all species  
in wash buffer for several days and the sections came out fine.

Good luck

Eric



__________________________
Eric Borneman
Dept. of Biology and Biochemistry
University of Houston
Science and Research Bldg. II
4800 Calhoun Rd.
Houston, TX 77204-5001




On Jul 23, 2007, at 10:53 AM, Szmant, Alina wrote:

> Hi All:
>
>
>
> We will be fixing some coral larvae and tissue for EM later this  
> summer
> and would appreciate any advice on good stopping places in the  
> fixation
> sequence:  We will be fixing with 2.5 % glut in Millonig's buffer, and
> post fix with 2 % osmium in same buffer.  We can't go all the way to
> embedding in the field but can take it though the osmium step.  It  
> will
> then be a few weeks before the materials can be dehydrated and  
> embedded
> in Spurr's.  So the question is:  where is the best place to stop the
> process in the field?
>
>
>
> a)       after the glutaraldehye (and whether to store in glut  
> sol'n or
> in the PO4 buffer?)
>
>
>
> b)       after the osmium step (in buffer or 70 % EtOH??)
>
>
>
> Thank you for sharing any tips you've developed over the years.  It's
> been awhile since I've used these methods and I imagine better  
> protocols
> for coral tissues have been worked out.
>
>
>
> Alina Szmant
>
>
>
> P.S.  EMS has a new, more expensive low viscosity 2 part Spurr's like
> medium:  has anyone tried it?  3 X the price but looks much more
> convenient.
>
>
>
> *******************************************************************
>
> Dr. Alina M. Szmant
>
> Coral Reef Research Group
>
> UNCW-Center for Marine Science
>
> 5600 Marvin K. Moss Ln
>
> Wilmington NC 28409
>
> Tel: (910)962-2362 & Fax:  (910)962-2410
>
> Cell:  (910)200-3913
>
> email:  szmanta at uncw.edu
>
> Web Page:  http://people.uncw.edu/szmanta
>
> ******************************************************************
>
>
>
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