[Histonet] RE: zinc fixative

gayle callis gayle.callis <@t> bresnan.net
Mon Sep 10 12:02:44 CDT 2012



You wrote: 


A wonderful colleague recommended to aid in my response that I add the
components I am using for preparation to assist in obtaining a more specific

response, about shelf life, so here it is: 


0.1   M Tris/HCl Buffer pH 7.4 - to which is added:


Calcium Acetate - 0.5 gm


Zinc Acetate - 5.0 gm


Zinc Chloride - 5.0 gm


The resulting solution was pH 5.45


According to my protocol my pH should be between 6.5 & 7.0 which is working.
I am just starting to work on developing this protocol and my Zinc Chloride
looked pretty pathetic in the bottle and I will be ordering fresh.  I think
this may have affected the pH - uncertain however at this point.



Invitrogen/BD Biosciences sells this fixative aka IHC Zinc Fixative
(formalin free) and are only vendors in the USA as far as I know although it
is sold in Europe under another name.   I did not find a shelf life in
either MSDS or Technical Data sheet, but you might want to contact them
about this.  When we tried this, we made it fresh.  You may want to look at
cost of buying the ready made compared to buying new chemicals or taking the
time to make it up - whatever is cost effective.    If you make it up, I
suggest using only fresh chemicals if any of yours  have been sitting around
on the shelf as we ran into the same problem with zinc chloride going bad.
The commercial IHC Zinc Fixative is a 10X solution (storage is RT) and
diluted with distilled water just before use - very handy.    


If you are interested, I have many publications on file about this fixative
including the original Beckstead (for human CD markers) and Nitta (for
murine CD markers) articles and would be happy to send these to you
privately.   At least the TRIS buffer can be made up ahead of time, maybe
even as a 10X solution and dilute when needed to add dry chemicals just
before use.   One thing is that the pH is never adjusted after adding the
chemicals to the pH 7.4 TRIS buffer.   This was mentioned in the original


Some things to think about when using this fixative.    Make sure your
tissues are not overly large/thick by reducing the sample size to achieve
total fixation since the time of fixation is limited.   BD Bioscience says
up to 48 hours.  I think one could perfuse nicely with this fixative too or
at least inject it into lumens, fill lungs, hearts, etc.   If you have
incomplete fixation with ZnTRIS buffer (Beckstead's fixative), then alcohol
during processing will complete the fixation which is something you do not
want to happen.    Nitta et al had a processing schedule in their
publication but we found we had to use shorter processing schedule for
murine tissues which became too dry and friable leading to poor microtomy
with overly long soaking to get a section.   Also the first NBF station on
the processor should be replaced with this fixative.  When working with
formalin sensitive CD markers which is the purpose of this fixative, I
wouldn't want a sniff of formalin from  NBF carry over into ANY of the
solvents.   Others may have more suggestions on this.      


I am not sure what you are using zinc fixative for, but presume it is for CD


Good luck, 


Gayle Callis


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