Re: Hard Tissue Histology

From:Mary Stevens <>

Hi Joan,

Fixation in 70% alcohol 1 week, or you can use 10%NBF but wash the specimens before processing.  If your animals were labeled with in vivo fluorochomes, it's been reported that the labels will fade with NBF (not something I've seen, but be aware.)  Prep the bones in the frontal plane, and cut off the distal ends.  Specimens measure about 1cm from epiphysis to the diaphysis (of course, this is assuming your area of interest is the metaphyseal/growth plate region.  We use methylmethacrylate (MMA) embedding and section on the Leica Polycuts with a carbon tungsten knife for all undecalcified specimens.  Rat tibiae may be processed on an automatic processor - in a 12 hours cycle start in 70% alcohol upto xylene.  The specimens come off in xylene and are placed into MMA I overnight at RT (80%MMA/20% dibutyl pthalate (DBT), then transferred to MMA II overnight at RT (MMA I + 0.2% perkadox-16, or you can use 99%pure benzyol peroxide  (BPO) at 2 or 3% concentration in your total volume).  Use vacuum for MMA I and  II (pull and relase 2 or 3 times, 15 mintues +/- each pull).  Rat Tibiae are embedded in 20mL scintillation vials in MMA III (MMA I + 0.4 or 0.5% Perkadox-16 or 4 or 5% BPO).  Tightly cap vials and allow them to polymerize on the bench top.  Polymerization usually takes 3-5 days, depending on the catalyst used.  Once polymerized (check under the hood with a probe to ensure the MMA is not soft or stringy), place the vials (with caps on) in a plastic bag and place at -20C for ~30 minutes or -80C for 15 minutes or so (times are not critical,  wouldn't leave the vials at -80 for more than an hour or 2.  Use a hammer ot crack the vials to remove the blocks from the glass.  I usually double glove and double bag so I don't cut myself on the glass peices (Others use molds or some type of plastic vials and cut the blocks out).  Place blocks into a RT water bath, run under water, remove from water, and leave blocks under the hood to air out at least overnight (put the blocks bone side down, since the last to polymerize is the top portion just incase some are sticky, plus to air them out better.

There are MMA kits on the market - which for us are expensive as we have a high volume of MMA work.  If you are only doing a few blocks, or if MMA processing will be occassional, I'd recommend a kit, hopefully others will fill you in on what kits they use.  All our solutions are from Fisher, except the perkadox-16 which we buy from AKZO Chemical.  It can be difficult to obtain depending on where in the US you are (I know in the west/midwest people have a hard time obtaining it.)  When using any type of MMA/DBT and catalyst be aware of it's hazards, you'll definately need to be in a good chemical fume hood with the correct gloves.  We use 4H silver gloves and cover them with nitrile.  Change the nitrile gloves when/if you come in contact with the solutions.  When mixing the solutions on the day of use (not ahead of time) let them stir, covered, in the hood for about an hour.  Use all solutions the same day you make them (you can store the solutions at 4C, but keep in mind that when they come out or the frig, MMA II and III may polymerize prematurely as they acclimate to RT, also be sure to allow all solutions to reach RT before opeing the bottle to avoid getting moisture into the MMA.  If your MMA is not crystal clear after polymerization - water has been introduced.

For Rat Tibiae, times breakdown as such:
Processing overnight
MMA I overnight
MMA II overnight
MMA III to polymerize 3-5 days
Crack out/air out blocks overnight
sectioning 5-15 minutes per block
Staining - deplasticize in 2-methoxyethyl acetate 4 changes at 15 minutes each, or some use xylene overnight.

That's about it - sounds simple, but anyone who's used MMA for undecalcified bone will tell you it's not simple until you've done it for a while, made all the mistakes that can happen (and there are quite a few.....).  The procedures will seem cumbersome and tricky, the chemicals nasty, and the time lengthy compared to paraffin.  Before doing any study bones, run the procedures on some "play" bones, i.e. non-study specimens.

Good luck.  

Mary Stevens
Genetics Institute
Andover, MA

>>> Joan Yonchek <> 09/26 2:25 PM >>>

I am interested in hearing from anyone currently sectioning
calcified, plastic embedded rat tibia's.  Specifically, I would
like to know the system you are using for sectioning,
your preference of embedding material and the time needed to
produce a stained slide.


Regeneration Technologies, Inc

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