Processing Calcified Bone

From:"Jacqueline D. Garfield"

Dear Histonetters,

I'm in search of processing protocols for calcified bone.   The samples for
this particular study are calcified sheep bone with tendon attached.  The
average sample size is 1cm X 1cm X 0.3mm.  Also, these samples have been
labeled with tetracycline dye.  

We are currently trying three different fixatives (NBF, Alcohol Formalin,
and 70% ETOH).   We plan on dehydrating in 70%, 95%, and 100% ETOH, for
about 4 days each; followed by xylene for 24hours.  

Then we have a dilemma?  What embedding media should we infiltrate and embed
with?  PMMA?  MMA?  or  JB-4?   And for how long?  I am having difficulty
finding protocols, especially for the PMMA method.  

Once embedded, we hope to be able to obtain 5  sections on a Leica RM 2155
microtome with a D-profile Tungsten Carbide Knife.  

Are we dreaming?  Or does this sound somewhat reasonable?  Your thoughts,
advise, prayers, and protocols are greatly appreciate.  

Thanks in advance,
Jackie Garfield




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