Re: Mineralised Bone biopsies

From:rueggp

Mixing your own Gylcolmethacrylate
Procedure from Patsy Ruegg

(A&C)  GMA Infiltrating Solution


1.    Measure 400 ml of 2-hydroxyethyl methacrylate GMA (HEMA) stored at 4d C

2.      Add 120 ml ethylene glycol monomethyl ether (EGME) (also called
         2-butoxyethanol of cellosolve) this is the plasticizer, the more you add the

         softer it is, too much can lead to a rubber eraser type consistancy which is

         very difficult to cut, too little will lead to very hard brittle blocks
which can
         shatter, play with this to get the desired hardness for your tissue sample
and
         enviromental conditions (high humidity can lead to soft blocks because the
         GMA will take on water, in very humid areas you might want to store the
         blocks desicated.

3.      Add 9 gm benzoyl peroxide

4.     Stir all together using a stir bar on a stir plate (takes about 20 minutes or
so to
         dissolve).
5.      Store completed, labeled and dated mixture in the refrigerator for up to 4
         weeks.


Infiltration is the most important step in obtaining good GMS sections. The samples
should be opened up if whole bones (femor, tibia, etc.) and sectioned to
1 mm in thickness if possible using a diamond wire or disc saw.
Infiltration should be done on a platform shaker (4 C) for as long as possible. The
longer the better. Several changes of the infiltration solution are recommended.
Biopsies are well infiltrated in 12 hours, larger samples of bone should be
infiltrated for several days. (week/weeks).



                Solutions

                2-hydroxyethyl methacrylate---Purchased from Polysciences-
                Catalog # 03699-0 Low AC*  CR2D2
                Cost approximately  $600.00   for 5 gallons (Store in refrigerator at
4dC).

                Ethylene gylcol monoethyl ether (EGME) Also called 2-butoxyethanol or

                cellosolve Purchased from Fisher----Catalog # E179-4

                Benzoyl Peroxide Purchased from Aldrich Chemical CO.-Catalog #
17,998-1


John Baker wrote:

> Hi Patsy,  Would you share your formula for making your GMA?  thanks,  John
>
> >Andrew,
> >
> >I would like to reinterate that GMA can be a good resin of choice
> >for Mineralized
> >Bone Biopsy work.  I find GMA to be easier to incorporate into a routine histo
> >lab than PMMA.  It is not as volatile and therefore less toxic when
> >used in less
> >than adequate ventilation or chemical fume hoods.  Although it is
> >not the resin
> >of choice for IHC because it cannot be removed like PMMA, you may not be doing
> >IHC on the mineralized bone samples anyway.  Most people are doing mineralized
> >bone histology to evaluate bone formation features for Metabolic Bone Disease
> >studies, such as Osteoporosis, Osteomalacia and Aluminum toxicity.
> >GMA will work
> >well for these studies including fluorchrome label evaluation and
> >histomorphometery measurements.  I think GMA has gotten a bad rap in the past
> >because people have used the embedding kits (JB4) which do not allow for
> >manipulation of resin hardness.  By making GMA yourself you will not
> >only make it
> >much, much cheaper, but also be able to control how hard it sets up for your
> >needs.  It is extremely easy to make up on your own.  This is my
> >nearly 30 years
> >worth of experience and plug for GMA for mineralized bone work.  You can do
> >fairly large, dense mineralized bone samples in GMA, I think very easily in a
> >routine histo lab.
> >Patsy Ruegg
> >
> >Gayle Callis wrote:
> >
> >>  You can make up your own polymethylmethacrylate (PMMA, and preferred by
> >>  many for bone, microtomed sections where PMMA can be removed for
> >>  immunostaining, ie Neil Hand protocols).  You need tungsten carbide tipped
> >>  blades, d profile, there are c profile available, d is excellent and a
> >>  microtome that will be strong enough to do the cutting, Olympus has one (do
> >>  not recall model) Leica 2155 and 2165.  As for a good sledge for
> >>  mineralized bone in PMMA, a Polycut, but it is pricey as are all
> >>  microtomes/knives for these protocols.
> >>
> >>  It is cheaper to not use kits, and you can use PMMA methods where washing
> >>  inhibitor away is avoided, getting rid of tedious, messy, handling of toxic
> >>  substance is reduced.  You will need a fume hood vented to outside, a
> >>  refrigerator, waterbath for polymerization.  Not sure if Technovits has a
> >>  PMMA kit suitable for microtoming, if so, it would be worth a try.
> >>
> >>  You can use an automated processor for dehydration and clearing, but not
> >>  infiltration with PMMA, use to vacuum dessicators for hands on infiltration
> >>  with PMMA.  The polymerization is best done in a waterbath for even
> >>  heating, ovens do not provide even heating with this step.
> >>
> >>  There are many publications in J of Histotechnology concerning these
> >>  methods, including Hand's antigen retrieval, look for these authors, Diane
> >>  Sterchi, J. Eurell, Cathy Sanderson (now Mayton), Chappell (hope I spelled
> >>  that correctly).
> >>
> >>  Working with undecalcified bone samples is more time consuming, there are
> >>  no STAT protocols, or you end up with a mess of bubbly, polymerized goo.
> >>
> >>
> >>
> >>  At 03:37 PM 2/27/02 +1100, you wrote:
> >>  >Hi Histonetters,
> >>  >
> >>  >We are in the process of setting up a Mineralised Bone Histo section in our
> >>  >Department and we need some advice. We are continuing the work of another
> >>  >department who have handed over this job to us and would like to streamline
> >>  >the process a bit. The way they make up their resin is long, tedious and
> >>  >tricky, so that is the first thing I would like to "fix". Secondly we use
> >>  >"spare time" on another departments' old sledge microtome to cut the bones
> >>  >and I would like to purchase our own equipment to get away from having to
> >>  >fit in with another lab.
> >>  >So firstly, has anyone had any experience cutting Mineralised (not
> >>  >decalcified!) bone on a heavy duty rotary microtome or should we be using a
> >>  >sledge?
> >>  >Secondly, what resins do you use and are they available in easy to use kit
> >>  >forms?
> >>  >
> >>  >Thanks for any help in advance
> >>  >
> >>  >Andrew Kennedy
> >>  >
> >>  >Senior Science Officer in charge - Histopathology.
> >  > >Department of Anatomical Pathology
> >>  >Concord Repatriation General Hospital
> >>  >Hospital Road
> >>  >Concord, NSW, Australia
> >>  >2139
> >>  >Phone: +612 9767 6115
> >>  >Fax: +612 9767 8427
> >>  >
> >>  >"Noli illegitimi carborundum"
> >>  >
> >>  >----------------------------------------------------------------------------
> >>  >----------------------------------
> >>  >This message is intended for the addressee(s) named and may contain
> >>  >confidential information. If you are not an intended recipient, please
> >>  >delete this email and notify the sender. Views expressed in this message
> >>  >are those of the individual sender and are not necessarily the views of
> >>  >Central Sydney Area Health Service.
> >>  >----------------------------------------------------------------------------
> >>  >----------------------------------
> >>  >
> >>  >
> >>  >
> >>  >
> >>  Gayle Callis
> >>  MT,HT,HTL(ASCP)
> >>  Histopathology Supervisor
> >>  Veterinary Molecular Biology - Marsh Lab
> >>  Montana State University - Bozeman
> >>  19th and Lincoln St
> >>  Bozeman MT 59717-3610
> >>
> >>  406 994-6367
> >>  406 994-4303 (FAX)
>
> --
> John A. Baker
> The University of Michigan
> Orthopaedic Research Laboratories
> Histology Unit
> 400 North Ingalls, G160
> Ann Arbor, MI 48109-0486
> Main lab office phone:734-763-9674
> Histology office:734-936-1635






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