rat tibia

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From:Gayle Callis <uvsgc@msu.oscs.montana.edu> (by way of histonet)
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A whole rat tibia is not difficult to decalcify, process or cut.

1.  Formalin will work best for cellular morphology, even if you decide to
do plastic, but you want paraffin, fix for more than a day or so, rodent
bone is dense, and change NBF after a day or so.

2.  Decalcify in 10% formic, or 4% formic/4% HCl, or other commercially
available decalcifiers.  Do endpoint checks to protect the cells, do you have
an x ray machine, Faxitron?  if so, use it for decal check, or do chemical
check.  Tibia will probably take a day and a half with HCl based, can be longer
Formic is a tidge longer, and you can start d'cal at end of day, go overnight,
then test next morning, change to fresh acid solution after a brief rinse,
continue during day, you may need to test later in day.  Whatever you do is
don't let bones go overnight in acid, even formic, if you are NEAR endpoint.
This can ruin the cellular morphology you want to see.

A big help is the Shandon nylon bags, one per tibia, use in processing also,
I suspend bone/bag in decalcifier, with xray test you can put 30 bones in a
big container, very efficient.  I use the little sales tags with string
and cut a slit at top of bag, put string through, loop over, hang this on
a pipette to suspend.  When you work with a huge volume of acid, VWR has
some polyethylene beakers with handles, much easier lift, don't have to
worry about breaking a big container.  They are cheap, up to 5 liter size.
We did 30 to 40 rat knees at a time, without any problems.

Just rinse the acid off, put back in NBF to interrupt decalcification, rinse
and resume decalcification the next day in fresh acid solution.

3.  When bones are decalcified, rinse with water for 30 - 60  min, process
in an automatic processor for 2 hours per station, starting with 70%, 80%
95% X 2, 100 %, xylene 1 change, Propar or Clearite 3 1 change, and
3 changes of paraffin.  Tissue Prep 2, from Fisher is excellent for bone,
a bit harder, and supports bone better during sectioning.  Vacuum and pressure
is on for all stations, and NO temps are added, ambient for solvents.

4.  Embed the same way for each bone, you can use peelaway molds, they are
deep, and push a regular cassette down into the back with lots of paraffin,
then the block will fit in microtome.

Have done whole rat femurs, whole rat knees  with this protocol, no problems,
but the real kicker is to do the endpoint test, that is done faithfully and
resulted in excellent morphology and could even cut at 3 micrometers.

Disposable blades work well, prefer high profiles since they are a bit
sturdier, others work well if you make proper adjustments to section.

As for histomorphometric anaylysis, there was discussion the last two weeks
on what was used.  John Tarpley is a good source for this, I am sure he has
done paraffin/bone/analysis.  Others also.

Good luck,
Gayle Callis

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