[Histonet] Trouble shooting decalcified bone sections (paraffinembedded)

[Jeanne Estabel]

Hi YiJing, 

We are collecting knee joint from mouse and we decalcify in 10% EDTA
(pH7.4).
Place the pots on a rocking platform under gentle agitation for 72 hours
at room temperature.
Then change the EDTA solution and place the pots on the rocking platform
under gentle agitation for a further 72 hours. The tissues can stay in
EDTA for 6 to 7 days.

I agree with Gayle that the processing could be also the step to look
at.

Regards

Jeanne


Jeanne Estabel, PhD
Scientific Manager
MGP Histology Operations Manager
Wellcome Trust Sanger Institute
Cambridge UK



-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of gayle
callis
Sent: 13 September 2010 17:24
To: 'Kristen Lauing'; histonet <@t> lists.utsouthwestern.edu
Subject: RE: [Histonet] Trouble shooting decalcified bone sections
(paraffinembedded)

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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