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. **************************************************************************** **************************************************************************** **************************** Julie, 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 here. 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 HTL,HT,MT(ASCP) _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet