RE: Processing Calcified Bone

From:Linda Jenkins


You stated:

"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 u 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."

First of all, I'm glad to see that you are trying 3 different 
fixatives.  Most literature indicates that formalin removes bone 
labels;however, I haven't found this be to true.  Hope you publish your 
results somewhere.
Second, are you using an automatic processor?  If so, you can decrease your 
processing protocol.

Here is my protocol:

10% NBF1 hr40°Cyes
10% NBF1 hr40°Cyes
70% ETOH1 hr40°Cyes
80% ETOH10 hrs40°Cyes
95% ETOH5 hrs40°Cyes
95% ETOH10 hrs40°Cyes
100% ETOH12 hrs40°Cyes
100% ETOH30 hrs40°Cyes

Now, as to the choice of monomer...that depends on what stains you want to 
see.  IHC stains are usually performed on MMA sections.  MMA resins can be 
removed from microtomed sections.  JB4 is a commercial kit that is=20
basically the same as glycol methacrylate (GMA).  GMA is cheaper and=20
usually faster to work with.  I can send you protocols for both  or go to as they have procedures for both resins.

And, finally, are not dreaming as your Leica 2155 will cut=20
mineralized bone beautifully, especially with the tungsten carbide=20
blade.  I have even cut bone with the disposable stainless steel blade!

Best wishes!

Linda Jenkins, HT
Clemson University
Dept. of Bioengineering
Clemson, SC 29634-0905

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