decalcified human bone-processing schedule

From:Gayle Callis <>

You did not give sample measurements???? If the piece fills the cassette,
and contains a great deal of cortical bone, you could add some time, 3 mm
thick.  Too thin (less than 3 mm) results in the  "potato chip syndrome"
and when trimming or sectioning, bone will pop out of the paraffin block.  

You should have good processing on tiny pieces with a routine schedule
(about 1 hour per station) for soft tissues.  If the bone is cortical, you
may want to extend (at the end) paraffin infiltration, often the place
where a problem arises.  This insures the decalcified bone matrix is well
supported for microtoming.  I would not use heat during processing since
this lends to hardening.  One trick is to use xylene for the first clearing
step, then Clearite 3 or Propar for the second clearing. The bone will not
be excessively hard, and the color is restored.  Two xylene substitutes are
also acceptable. Tissue Prep 2 or another harder paraffin is nice for
embedding, much firmer support of bone and when used for infiltration - a
real bonus!  Preference is a paraffin with melting point of 56C, then I
infiltrate at 56C - 58C.  Our VIP is excellent for holding temperatures,
and can infiltrate at 56C! wonderful for bone. Be sure to use vacuum and

Nice job on decalcification ---- endpoint test prize of the day!!  My

At 02:32 PM 8/14/01 +0200, you wrote:
>Hi histonetters,
>I am processing small bits (to fit a histo. cassette) of human bone 
>which have tendon attached. I have decalcified in 2% nitric acid. (
>Checked endpoint.) I would like some advice on the best way to 
>process these pieces of bone plus attached tendon. I do not have much 
>experience working with bone samples and I have obtained a lot of 
>information off the histonet.  I feel that I should extend processing 
>times but I would like to know what is optimal without wasting too 
>much time. 
>Sharon Marshall
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology
Montana State Universit

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