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

[pblackwelder at rsmas.miami.edu]

Hi Alina,

May have a few words of advice here...if you are preparing samples in the
field I would suggest you stop in the initial fixative...and if it will be
a few weeks I would make changes to fresh fixative every week or
so...making sure your samples are always immersed...refrigeration would be
important if possible...samples left in buffer often result in microbial
contamination after a few days..especially if refrigeration is a
problem...the new resin you are referring to may be LR White?..tried it
once... it requires fewer steps than Spurr, but I'm older school and like
working with Spurr's...especially if you are cutting calcified
material...although heard from others that LR White works well...another
good source of info on this topic would be Walt Goldberg (and several
others as well)..any comments Walt?...good to chat with you Alina..been a
long time...Pat Blackwelder



> 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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