RE: Processing Calcified Bone
"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
Second, are you using an automatic processor? If so, you can decrease your
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
http://www.ebsciences.com/ as they have procedures for both resins.
And, finally, NO...you 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!
Linda Jenkins, HT
Dept. of Bioengineering
Clemson, SC 29634-0905
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