decalcified rat elbow samples

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From:Gayle Callis <>
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Several things.  This protocol is the same for mouse except processing time
is less per station and for rabbit, more time is necessary, usually we
bisected the rabbit joints to permit better fixation, speed up
decalcification AND processing.   

Work with a whole elbow joint, it is possible and not that difficult, no
more so than whole rat knees. 

Dissect excess muscle, skin away from OUTSIDE of joint carefully, do not
nick the cartilage.  The humerus and radius/ulna do not have to be left
whole, cut across these bones with a small saw above and below the joint do
not crush bones.

Fix in NBF for several days, rat joints left intact, need to fix longer
than 24 hours, a week for knees was ideal, and put into a large amount of
fixative, change after a day or so, 20 to 1 ratio, I like more, NBF is
cheap.  Bones must be totally fixed BEFORE you can decalcify in acids, or
you macerate the tissues.  Decalcify in 10 - 15% formic acid, or 4%
formic/4% HCl (fast!), there are commercial decalfifiers available, many
HCl are over 10%, a buffer formic acid with sodium formate or sodium
citrate has approx 4 -5 % formic acid concentration, and will decalcify
slowly.  Change decalcifier daily, and after each endpoint determination.
Rinse bones between changes, to remove any calcium residing on surface of
bone. Do endpoint determinations to insure complete decalcification AND
avoid the problem of overexposure to acids aka "overdecalcification".
Bones should be suspended in the decalcifying solution using Shandon nylon
bags which can then be used for processing.  Tie the bag shut with string.
Suspenstion permits all around contact of decalcifying fluid to bone.  Do
not use heat, stirring can be used, but that is a debatable point.   

Rinse bones in running tap water for 1 hour or so, then process through 70,
80, 95 X 2, 100 X 2, xylene or xylene substitute, paraffin X 4 if possible,
or at least 3 changes of paraffin UNDER VACUUM, 2 1/2 hours per station.
Use a harder paraffin for infiltration and embedding if possible, better
sectioning in the end, Tissue Prep 2 works well from Fisher.  If not use
the harder paraffin for embedding to give good support during sectioning.
Hopefully you have an automated processor to do this, if not you will have
to make sure the bones are dehydrated correctly, carousel styles without
vacuum capability tend to require additional time.  Size of bones, age of
animal, whole or partial bones, type of bone (cortical vs trabecular) all
determine time needed for processing.  Try a test run to see if you get
results.  If your animals are age matched, you can usually estimate the
decalcification time AFTER you do one or two with endpoint test, and get
away from less testing IF you are diligent.  

Decide how you are going to orient the bone to see the articular surfaces
you want to see.  Embed whole bones in large peelaway molds, push the
LEVELING CAREFULLY.  When sectioning, you can then do semiserial sections
through the joint at 5 um, and if the cartilage tends to curl, one can add
a detergent (TWeen 20 or even DMSO) to the waterbath, do not DMSO with
metal pan, and keep skin out of it, work in a well ventilated area.  This
will allow wrinkles to smooth out, avoid high heat to spread out the
section or the cartilage can separate from harder bone, explode the
section.  Drain sections and dry FLAT at 37C overnight (minimum) or for
several days (I know a gal who dries at this temp for a week or longer)
before staining.  

When trimming the block, do it slowly, and not with thick slices, have been
known to use a NEW knife to trim, to prevent bone from catching and popping
out of block.  You can soak the trimmed block for a short time on an ice
block with water, then resume cutting.  Oversoaking tends to make the bone
swell out of block, and creates havoc with the cartilage in relationship to
the bone.

We were able to do several hundred whole rat knees this way, and maintain
the joint spaces with articular cartilage and synovium, with excellent mid
sagittal cuts through the growth plate.  Works for rabbit joints also.  

Don't panic, take your time, good luck, enjoy, it is a fun technic.

Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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