Decalcified bone, hard specimen, long reply

From:Gayle Callis

Debbie and Linda, 

The NSH-VIR animal tissue processing manual is almost ready for printing,
final corrections and refinements being done.  I will let everyone know
when this is available.   

As for the bone problems, you are not alone! Some things to help alleviate
hardening can be done.

Cutting bone slabs too thin creates a sample prone to "potato chip
syndrome", bone collagen matrix is extremely dense, even after
decalcification, then matrix is made even harder by alcohol dehydration and
clearing.  For latter, a xylene in first station, followed by Clearite 3 or
Propar station helps, or two xylene subtitutes, followed by harder paraffin
infiltrations (Tissue Prep 2) embedment in same.  You want to try and match
the hardness of bone matrix as closely as possible with a harder paraffin.
This is why plastic is so successful with undecalcified bone. 

Also increase infiltration times, and keep infiltration temperature close
to melting point of paraffin, as heat contributes to hardening.  As for
potato chip problem, when block is trimmed, very thin bone slabs wants to
and will pop out of the block, keep you thickness at 3 mm minimum, same as
soft tissues, thinner and you have potato chip (there is little sample to
infiltrate and be held firmly in supporting paraffin).  If bone is dense
cortical bone, it may require longer processing.  An hour per station for
your samples is probably adequate for ambient temperature
dehydration/clearing, but longer paraffin infiltration - the slowest step
with denser melted paraffin, may help, or put bone in a vacuum oven for
longer infiltration while you embed your other samples.     

Sectioning with a sturdier knife helps, low profile knives tend to give me
more chatter - springing of blade (unless the bone is perfectly processed,
and not a great deal of cortical bone), so we use high profile and only
sharpest blades.  Accuedge blades are excellent, also Richard Allan Edge
Rite and DuraEdge are the favorites these days.  

BE sure you embed so a corner of bone (narrowest portion) is cut by the
knife first, this is the path of least resistance over the knife with
widest part of bone cut last,  and after a careful trim (I even use new
blades for this!), soak bone on an ice block with water for a short time,
and section with a brand new edge, carefully approaching knife without
wasting the soaking effects. If you oversoak bone, it will swell out of the
block, and may pop out when cutting - trim/cut carefully, not thick trimmings.

Good luck and you should see the dents in my walls, also in walls across
the hallway!!!  not the least of which is unrefined bad language habits - I
supply ear plugs to those who protest. 


At 09:50 AM 1/30/02 -0500, you wrote:
>	This is a common problem with decals.  Modification of the processing 
>protocol is necessary to keep specimens from becoming almost as hard as 
>before decalcification.  Gayle Callis and Diane Sterchi have done many 
>workshops on this subject and have just recently edited a processing manual 
>for the VIR committee ( Gayle, is this available for purchase, yet?).  I 
>think there may be one or two processing protocols in there that may help.
>	If all else fails, bounce it off the wall...just kidding...although I've 
>been severely tempted by some of the decals I've had to cut.
>	Linda
>Linda Jenkins, HT
>Clemson University
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

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