[Histonet] RE: zinc fixative and decalcification with EDTA

gayle callis gayle.callis <@t> bresnan.net
Fri Sep 18 10:11:28 CDT 2009

You wrote: 


I have recently used zinc fixed tibia and calvaria for the staining of CD31.

My CD31 does seem to have worked, however there seems to be marrow loss and

digestion of the marrow also.  


I zinc fixed the bones for 48 hours and then used EDTA pH 7 for

decalcification for 2 weeks. I am unsure whether I have fixed the bones for

too long, whether the decal has been affected by the zinc fix (as the bones

were "jelly-like" before processing) or whether the decalcification needs

shortening when using zinc fixation.


If anyone has any experience in using zinc fixation on tibia for Immuno

staining and can offer some advice that would be much appreciated.





By zinc fixation, I presume this is the Beckstead formalin free Zinc TRIS
buffer mixture (ZSF)?  If so, what you may have done is reverse the fixation
by decalcifying the bone with EDTA rather than have the decal affected by
the fixation. EDTA will chelate the zinc, along with all the other metal
ions in bone e.g. calcium, magnesium, and iron.  This very well may affect
the condition of the bone after several weeks. I once again presume you are
working with mouse bone?  You did not provide some specific information


I have not seen publications using this fixative for bone followed by
decalcification, and it is a distinct possibility this will NOT work. I have
some excellent publications on using ZSF for soft tissues.  


We do all our murine CD marker staining on undecalcified bone frozen
sections after sectioning with Cryojane tape transfer system from
Instrumedics using a tungsten carbide knife.  


Here are two publication references, freely accessible on J Histochem
Cytochem to help you. I also have these in pdf and will send privately.


Kim L. Kusser and Troy D. Randall
Simultaneous Detection of EGFP and Cell Surface Markers by Fluorescence
Microscopy in Lymphoid Tissues
J. Histochem. Cytochem., Jan 2003; 51: 5 - 14.


S Mori, T Sawai, T Teshima, and M Kyogoku
A new decalcifying technique for immunohistochemical studies of calcified
tissue, especially applicable to cell surface marker demonstration
J. Histochem. Cytochem., Jan 1988; 36: 111 - 114.


Gayle M. Callis






More information about the Histonet mailing list