Hello Efrain and the rest of histo-land

Below is the method I use for dealing with bone and immunos, and bone
adhesion in general for other special stains. This technique has been
working well for us, and may or may not work for your specific antibodies.
Most of this has been discovered through trial and error (and error, and
error...).  I've been through almost everything out there, for immunos, in
situs, and other stains on calcified and decalcified bone.  You can also
check the histonet archives on the bone IHC subject, and find even more
advice.  The archive address is www.histosearch.com/histonet.html

I use APES (Sigma cat #A-3648) coated slides, prepared in house (pre-rinse
slides in acetone, 2% APES in acetone for 30 sec, rinse in acetone, wash in
running dH20, air dry overnight).  I then cut 4-5um sections, collect onto
the coated slides, and bake them flat for a couple of hours in a 60C oven.
After dewaxing and running down to water, I then bake them flat again at 60C
until they are dry, generally 30-45 mins. For epitope retrieval, I've been
using Dako citrate solution, heated to 70-75C in a coplin jar in a water
bath.  I maintain the slides in the retreival solution for ~40 mins, remove
the jar from the water bath to the counter top, and allow it to cool for
another ~20mins.  I've found that I lose most of the sections if I heat them
to 95C as Dako recommends, and microwave retrieval peels off the cortical
bone instantly.  This gentle heat works in most cases; for more difficult
antigens, I follow the heat step with either pepsin or proteinase K,
anywhere from 25-100% of normal working strength.

I wash gently between immuno steps, and lay the slides flat to air dry after
counterstaining, instead of running them down through alcohols.  I generally
don't xylene dip them before coverslipping- yes, I get a few bubbles, but
don't want to risk losing the tissue after all of the rest of the work is
completed.

In most cases, the cortical bone will remain attached, sometimes I get some
peeling on the outer edges.  The trabecular bone, cartilage, and marrow
generally remain intact throughout this procedure- on occasion I over
digest, and lose some of the marrow.  You'll have to try out a few things,
and work it through for your antibodies of interest.

Hope this helps

-Mike


Michael Archambault, Research Scientist
Bone and Soft Tissue Program
Osiris Therapeutics, Baltimore MD
410 522-5005 x 226
marchambault@osiristx.com

> ----------
> From: 	Efrain Pacheco[SMTP:epacheco2001@yahoo.com]
> Sent: 	Thursday, August 03, 2000 5:07 PM
> To: 	Histonet
> 
> Hi,
> First question: Does anybody had any experience doing
> Heat Induced Epitope Retreival (HIER) on bone,
> particularly rat femurs? We have two antibodies that
> react well after HIER except for the fact that the
> cortical bone tends to fall off the slide, definitely
> not good. 
> Second question: does anybody know of an antibody that
> identify osteoclasts on fixed paraffin tissue? Of
> course, preferably one that do not requires HIER
> pretreatment, for reasons discussed on the first
> question.
> I will appreciate any suggestions.
> 
> Efrain Pacheco, HT (ASCP)
> Histology Associate
> Amgen Inc.
> 
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