Re: Perfusing with zinc salt fixative

From:Gayle Callis

At 12:19 PM 7/24/2003 -0700, you wrote:
>Hi again,   I apologize for not giving the recipe for the fixative I was
>using. It is the zinc salt fixative from the 1994 Beckstead paper (J.
>Histochem Cytochem 42: 1127-1134). The composition is as follows: 0.1M
>TrisHCl (pH 7.4) with 0.05% Ca acetate, 0.5% Zn acetate and 0.5% Zn
>chloride.  The final pH of this fixative ends up being around 6.8, but you
>are warned not to try to bring it closer to neutral pH as precipitates will
>form. Initially I tried clearing the blood from the mouse with 0.1M
>phosphate buffer. However, as soon as I started running the zinc fixative
>through the tubing (i.e. before it even got into the animal), a white
>precipitate formed in the tubing - and i imagine also in the animal. I
>assume this was due to the zinc fixative coming into contact with phosphate
>molecules left in the tubing from the previously run phosphate buffer. I
>next tried clearing the animal with 0.1M Tris base buffer, pH7.4. I did not
>see any precipitation form in the tubing, and have no idea what was
>happening in the actual animal. I wasn't sure though if the Tris was a good
>buffer with which to clear out the blood in the animal. So, my questions
>are: 1). Is this zinc salt fixative a good fixative for doing whole animal
>perfusions? 2). If it is, what buffer would you suggest for clearing the
>animal with prior to fixation 3). How long would you post-fix an adult
>mouse brain for, and would you recommend trimming the brain to the smallest
>possible size (i.e. only taking the part in which I am interested) so as to
>decrease the time needed for post-fixation?  II molecules in the mouse
>brain. Pharmingen recommends the use of a non-formalin, zinc fixative as
>the MHC molecules are known to be difficult to localize with
>immunohistochemistry in paraffin embedded tissue  

Your observations on using Zinc Fixative perfusion are interesting. We
prefer to call it ZnTRIS fixative to distinguish it from zinc formalin.    

As for a buffer rinse, it may be worthhwhile try TRIS buffered saline (TBS,
the formulation used for immunohistochemical staining) containing with
heparin first THEN run ZnTRIS fixative. Having the saline in the TRIS
buffer before ZnTRIS may help prevent ppt of salts you observed, or was it
the zinc salts coming out of solution?? It may be that TRIS is incompatible
for perfusion, I don't recall ever seeing it used.  Post fixation should
stay within limits of recommended fixation (Nitta et al) of 72 hours.  

If that doesn't work, why not fresh unfixed mouse brain snap frozen rapidly
(we used a dry ice/isopentane slurry) then crysosectioned.  We just
cryosectioned snap frozen fresh mouse brains at -17C, and have wonderful
frozen sections.  Our CD markers worked perfectly on acetone/alcohol fixed
frozen sections (excellent morphology), plus 50 sections/3 different
antibodies were done in a morning.  Frozen sections are not all bad and
fast! - particularly with brain/murine cell surface marker work. Cutting
paraffin sections after fixation with ZnTRIS was not very good.  Tissues
were dry, friable, and morphology touted as being good with with fixative
IS not comparable to NBF fixed tissues.   IF you do not have total ZnTRIS
fixation, the processing alcohols will complete fixation and this may not
be optimal for your MHC II markers either. We tried fixative and opted to
go back to our beloved frozen sections, far less work. 

The publication by Beckstead was using this fixative for human markers, not
murine.  Nitta et al in Cell Vision, vol 4, #1, February 1997 did the
murine lymphocyte cell surface markers work using this fixative with
immersion technic.  When we tried it, all fresh tissues were reduced in
size for rapid, total fixation via immersion. I suggest you not do
perfusion but reduce size of brain for area you are interested in, and do
immersion fixation if TBS/heparin does not work, you should still have good
fixation without perfusion.  

A tough call, let us know if you try TBS/heparin.


Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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