[Histonet] Re: rabbit bone specimens

From:Gayle Callis

Some questions first:

Are you working with whole femurs and tibias OR do you bisect the 
samples?  Cutting into slabs is also possible using a diamod cutoff blade 
i.e. Buehler Isomet.  This should be done BEFORE fixation to speeds up 
fixation and eventually speed up decalcification by simple size reduction. 
There was an excellent article in the old Stain Technology about working 
with rabbit bones some years back.  Ohrt, C. et al. Preparation of whole 
rabbit knee joints for microscopy examination, Stain Technology, 
61(6):353-360.004, 1986.  This article is excellent.

You refer to Cal Ex decalcifier, but which one?  The dilute HCl Cal Ex is 
1.34 M HCl when calculated is 8.3% HCl and is rather 
strong!!  Decalcification in this solution will more than likely result in 
overexposure to acid aka overdecalcification, and will damge the bone, and 
nuclei in all cell types.  Bones must be totally fixed before going into 
any acid, so fix whole bones for a week or longer, and change fixative 
after 1st or second day to replenish fixing agent.

If the Cal-Ex is the fixative/formic acid combination, check the MSDS as 
the formic acid may be close to 10%?  Two weeks in this solution, if a 
whole bone, may result in some autolysis, as this decalcifier is designed 
for tiny bone biopsies.   It would be better to fix in formalin then 
decalcify in a formic acid decalcifier, particularly for that period of 

Do you do decalcification endpoint determinations?  If not, 2 weeks in 
either of these decalcifiers could result in overexposure to acids and 
damage bone, soft tissues.  Formic acid can be just as damaging to tissues 
if the bones are left in too long.

Rabbit bone can be difficult since the articular cartilage is thinner than 
some species.  When you process for longer period of time, the cartilage 
becomes very dry since water bound to protein in cartilage is removed along 
with free water, not ideal.  The bone however, tends to be ok due to its 
density but needs the longer processing for just that reason, in order to 
dehydrate, clear and infiltrate.

How to compensate:  After facing the block,  lay block on an ice block with 
water on top for a few minutes, NOT a long time or tissue will swell out of 
block.  The section should be very flat, without compression as it comes 
off the blade edge.   Using Tissue Prep 2, a hard paraffin helps support 
bone with cartilage, or some other harder paraffin.

IF your cartilage continues to look wrinkled or did not flatten out on 
water bath, you can try several things.  One, lay section on cold 10% 
alcohol, pick up and go to warm water bath to let it flatten - be careful 
the sections are not exploding, so use cooler waterbath i.e 
41C.    Second:  try Tween 20 (not sure of concentration, others have used 
this) in the waterbath, not a great deal, but just enough to break surface 
tension on water, and let section flatten then pick up.  Third:  use a 
waterbath that is 5% DMSO (not great to put hands in or breathe) but this 
will flatten cartilage wonderfully.

Perfectly flat cartilage on slide surface is crucial for section adherence 
to slide surface.  Some gelatin sub slides (don't do this to pricey plus 
charge, but presub clean, regular slides, then pick up sections onto these).

When you dry slide, do it at 37C to 40C keeping slides FLAT after a blot 
and good vertical draining.  Dry overnight but longer is preferred here - 
several days.  Drying bone w/cartilage sections at 60C continues to dry out 
cartilage even more, and when you stain, the section releases and curls 
up.  BEWARE of harsh running tap water rinsing!!  Gentle water rinsing 
only, and do not use ammonia water as bluing agent, it may cause section to 

As for processing, we did 1 1/2 hours for thinner slabs (4 -5 mm) and 
bisected in half knees for 2 hours per station with minimum of 3 paraffin 
changes always with vacuum and pressure.  A popular schedule here is 70%, 
80%, 95% x 2, 100% x 2, xylene x 1, Clearite 3 x 1, paraffin X 3 (we do 4 
changes).   The xylene in first change insures clearing with no residual, 
and Clearite 3 is less hardening to bone.  You can use Propar and you can 
do two changes of these substitutes, have tried them all successfully with 
larger bone.

Use the best, sharpest blades possible, high profiles are sturdier and just 
as sharp as low profile, but cut dense bone better without chatter 
problems.  Change to new sharp edge frequently, this is often done between 
ribbons here and even and blocks.  That sharp edge is a must.

The bunny can be more difficult, but good sections are possible.  Just try 
some of suggested tweaks.

Vyou wrote:
>I am working on a research projects with rabbit femurs and tibias.  They 
>are decalcified in cal-ex from fisher for 2 weeks and then processed for 
>1.5 hours in each solution on the processor.  2 changes of 70, 95, 100%, 
>xylene and then paraffin.  I am really having problems with the cartilage 
>folding right at the curve of the specimen.  It seems as though the bone 
>just below the cartilage is not thoroughly processed or  hard. When I do 
>feel I get a decent section it seems to fold when I stain.  I am using 
>plus slides. I don't normally do a lot of research so if anyone can help 
>me with this problem either with cutting or processing solutions I would 
>really appreciate the help.
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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