[Histonet] Fixation for EM

[Va Paula Sicurello]

Hi Chana,

I'm going to highlight my answers after each of your questions below.

Though I will say that you should contact whomever may be processing the brains for EM to see what they would prefer.  Also ask them which buffer they prefer.  Karnovsky's fix is traditionally made with Sodium cacodylate (an arsenic base compound) but some labs have switched to phosphate buffer for safety reasons.

If you have any other questions, please feel free to contact me.

Paula  :-)

Paula Sicurello
VA Medical Center San Diego
Veterans Medical Research Foundation (VMRF) 
Core Research Imaging Center
3350 La Jolla Village Dr., MC151
San Diego, CA 92161
858-552-8585 x2397

http://www.vmrf.org/researchcenters/confocal/confocal.html



> 
> My questions follow:
> 
> (1) What is the best fixative solution to be used under
> the
> circumstances? I was thinking of using a combination of
> paraformaldehyde and acrolein due to acrolein's penetrative
> qualities
> and superior fixative strength. 

 I think most labs tend to stay away from acrolein (it is tear gas and not pleasant to be around).  A modified Karnovsky's is probably the best all around general EM fixative.  If it's OK to mince the brain into 1mm cubed bits that would be best for proper fixation.  If you can't then you end up with a bit of a fixation barrier.  Glutaraldehyde penetrates at 0.5mm/hour at room temp.  When the outside parts get fixed first and the sample is large, then fix for longer periods of time.  Samples can be left in the refrigerator for weeks.
> 
> (2) Do I need to perform a secondary fix with osmium
> tetroxide
> myself...or do I leave that to the EM lab techs to perform
> when they
> make slices? If at all possible, I want to minimize the
> amount of
> tissue processing on my end.

Unless you have to, I would avoid the Osmium textroxide.  A good EM lab would prefer that you just fix your samples and either submit the samples in buffer or fixative.  While acrolein is tear gas, Osmium textroxide is seriuosly poisonous.  Exposure to it can cause blindness and death.  With an exposure limit of 0.002ppm it is nasty.  The one good thing is you will go blind before you stop breathing and the blindness only lasts a few months.  So if you can't see or things get blurry, it's time to get some fresh air   ;-).  
    Seriously, let the EM people do the Osmication.

> 
> (3) How long can the brains be left in post-fixative
> solution prior to
> further processing in the EM lab? Or, in other words, is
> there a
> maximum time period after which fixed whole brains cannot
> be sliced,
> washed, embedded, or otherwise processed?

Theoretically, samples can be left in fix a long time.  I once forgot a monolayer of cells and left them in Karnovsky's for 2 weeks without any noticeable deterioration of the ultrastructure.  
Again, ask the EM people, some have protocols they like to follow and times they prefer for optimization of image quality.
> 
> (4) Any other suggestions, comments, etc.? Obviously, there
> has to be
> a less-than-ideal division of labor in this matter because
> we do not
> expect the EM lab to remove brains from rats that have been
> dead for
> up to 48 hours. I can handle that part, but I want to
> ensure I do
> everything right because what I do affects the rest of this
> study.

Most EM labs prefer that the user do a minimum of processing to the samples submitted.  We are a superstitious bunch and like to be able to control almost the entire embedding process.  That way if something gets messed up we can figure out what we might have done wrong and correct it.
> 
> Thank you for your time and any valuable insights into this
> matter.
> 
> Chana de Wolf
> Advanced Neural Biosciences, Inc.
>