Atoska,

This recommendation was for whole critters.  That's how retrograde tracing is done.  
You apply or inject the dye to a cut nerve ending and let it transport up through the 
axon to the cell soma.  Thus you can map the anatomy of any given nerve. This can be 
done on a live critter or it can be done on a fixed critter.  I assume Trisha was 
talking about fixed pigs.  And that some nonfixable dye such as DiI or a nuclear dye 
was being used.

When you use an injectable dye to inject into specific cells in a living creature, 
the dye has usually been modified by adding lysine or some other amine-containing 
moiety to it.  Thus, you can later fix the organism with an aldehyde fixative such as 
formalin or paraformaldehyde to crosslink the dye to components in the cell, and thus 
fix it in place.  Otherwise, the dye, which is a relatively small molecule, will 
diffuse out of the tissue.   This is true, regardless of whether you are doing 
paraffin or frozen sections.  Again, people tend to avoid formalin, as it can 
compromise the membrane before the dye is fixed in place.  It should be noted, if 
your tissue tends to autofluoresce, this tendency is often amplified in paraffin 
work.  If you're doing fluorescence work, you want to avoid using glutaraldehyde, as 
this causes the tissue to autofluoresce.

It's possible that your injectable dye is a high-affinity nuclear dye, in which case 
other types of fixation would work.  You really need to know a bit about the nature 
of the dye to determine which fixative to use and how to treat the tissue.  For 
instance, DiI, which is a commonly used neuronal tracer, is believed to incorporate 
in the membrane, and to passively transport through the membrane to the cell soma.  
When this dye is used, you need to avoid using detergents, alcohols, or organic 
solvents, as this will pull the dye out of the membrane.

Karen in Oregon




Date:          Mon, 6 Mar 2000 14:52:48 -0600 (CST)
To:            larisonk@uoneuro.uoregon.edu (Karen Larison)
From:          Atoska Gentry <gentras@vetmed.auburn.edu>
Subject:       Re: Paraform/fluoro


Is this a recommendation for paraffin sections, frozen sections , or both?
We need help in the area of frozen sections in which fluorescence has been
injected.  Is it safe to fix them and if so what fixative should we use?
Thanks, Atoska

Atoska S. Gentry B.S., HT(ASCP)
Research Assistant
Scott-Rtichey Research Center
College of Veterinary Medicine
Auburn University, AL  36849
PH (334) 844-5579
FAX (334) 844-5850


Date:          Fri, 03 Mar 2000 11:15:00 +0000
From:          larisonk@uoneuro.uoregon.edu (Karen Larison)
Subject:       Re: Paraform/fluoro
To:            HistoNet@pathology.swmed.edu

Trisha,

What fluorescent retrograde tracers is he using?  If he's using DiI, I would 
absolutely recommend sticking with aqueous-based fixatives, preferably 
paraformaldehyde.  Don't use Prefer if it contains alcohols or organic solvents!  
Other fluorescent tracers may be less picky, but I would still advise caution, 
because most neurotracers work because they can't breach the membrane once they are 
in the axon, so you don't want to use anything that can possibly perturb the 
membrane.  That's why people use paraformaldehyde rather than formaldehyde in these 
applications.  The methanol in formalin has the potential to perturb the membrane and 
you can get cross-talk between in axons in the nerve.  In otherwords, the dye crosses 
too easily from axon to axon instead of staying within the axon of interest.

Paraformaldehyde is no different than formaldehyde once it's in solution.  However, 
it will tend to polymerize over time, and precipitate.  Thus, you have to make it 
fresh.  The purpose of the methanol in formalin is to keep the formaldehyde in 
solution.  But it can and does mess up cell membranes.

Personally, I wouldn't ask him to venture into places unknown with unknown additives 
in the commercial fix.  It's just too likely to mess up his experiments.  And some of 
these tracer experiments are quite long and involved.  He should stick with the 
paraformaldehyde.

Karen Larison
Institute of Neuroscience
University of Oregon

 


Date:          Thu, 02 Mar 2000 18:46:52 -0800 (PST)
From:          "P. Emry" <emry@u.washington.edu>
Subject:       Paraform/fluoro
To:            HistoNet@pathology.swmed.edu

Hi all,

I am working with a post doc who wants to inject some "fluorescent
retrograde neurotracers" in to pigs and use paraformaldehyde to fix the
specimens. (Forgive me if I have muddled that up.)

He is being very kind in considering my formaldehyde aversions and 
said he would work after I go home.  He is reluctant to use my
substitute Prefer thinking it may effect the fluorescent signal.

Have any of you worked with Prefer or any other
formaldehyde-paraformaldehyde substitute for that use?  (I have never used
paraform. and have no idea how it differs from formaldehyde.)

I am sure he will want some references if possible.

Thanks,

Trisha