[Histonet] Trouble shooting decalcified bone sections (paraffin embedded)

[gayle callis]

YiJing, 

You could be under- processing the mouse paws.  We extend processing time
for mouse paws during dehydration, clearing and especially infiltration with
paraffin and done with vacuum. Poor paraffin infiltration can really create
shredding problems. There is a simple, cheap way to check endpoint of
decalcification when decalcifying bones.  It is called weight loss/weight
gain method and works for both EDTA and any acid decalcification protocols.
Endpoint determination is important to know when calcium is totally removed
so processing and microtomy problems do not occur.  Bending or "feeling
soft" generally is a poor way to determine total calcium removal as small
calcium deposits are not detectable this way.  X-ray is the most sensitive,
but not everyone has the technology/equipment for that. Chemical endpoint
testing is also simple and easy to do. I doubt the blades are the problem,
but more likely decalcification and processing. I would be happy to send the
weight loss/weight gain and chemical test methods to you.  

Using a harder paraffin helps with bone, Tissue Prep 2 (Thermo Scientific
under Fisher Scientific label) is one of several available although other
paraffins should work if infiltration is done properly. 

You did not provide a processing schedule for these decalcified mouse paws.
Is this done manually or on an automate processor?  Times in each solution?
Time of paraffin infiltration? 

Gayle M. Callis
HTL/HT/MT(ASCP)     
 

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Kristen
Lauing
Sent: Monday, September 13, 2010 9:13 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Trouble shooting decalcified bone sections (paraffin
embedded)

I section EDTA-decalcified mouse tibias frequently, and I find it helps if
the paraffin is very very cold during sectioning - if the tissue starts
shredding again, I press a large piece of ice to the block for a minute to
cool it down without having to remove the block from the microtome.  It
seems to work well for me, although it may not be the most technically sound
way to get the job done.  I can usually get a good ribbon of sections this
way when I'm having difficulty with a particular sample.
I have to use an old microtome and blades because I'm a student at a
research institution, without the help of professional histotechs.  Our
tibias are decalcified in 10% EDTA for 7 days at 4 degrees with agitation
with frequent solution changes.

Kristen

>>> "CHEN, YIJING" <ychen9 <@t> kent.edu> 09/12/10 12:50 AM >>>
Hi,
We are having difficulty sectioning paraffin-embedded decalcified adult
mouse autopods (paws).  The tissues shatter as soon as they hit the blade
when sectioned.  

The autopods were soaked in CalEX for 4 days at room temp and felt extremely
soft before embedding, suggesting effective decalcification.  We use the
Sturkey EXTREMUS low profile disposable knives on our microtome.  These
knives are said to be coated with the hardest nitride coating available.
Any suggestions will be greatly appreciated.
Sincerely,
Yijing


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