SPI-Chem™ "Low Acid" GMA Kit Hard for TEM Thin Sections
Use instructions
SPI-Chem™ Low-Acid GMA Water Soluble Embedding Kit for Transmission Electron Microscopy (TEM) Applications
Instructions for Use of SPI #02630-AB and SPI #02630-AA
CAUTION:
Thoroughly read all instructions and warnings on container labels prior to use of this kit.
Introduction:
The polar water soluble, 2-hydroxyethyl methacrylate, commonly known as "glycol methacrylate",
or GMA, has found an increasing number of applications for the embedding of biological tissue
for transmission electron microscopy (TEM), for the preservation and observation of fine
structure not previously subjected to solvent dehydration. GMA forms only non-crosslinked
straight chains on polymerization and therefore requires no hardener. The catalyst used to
promote end-to-end polymerization is benzoyl peroxide, at a concentration of about 0.6% in
the final infiltrating medium.
GMA embedments, and specifically low acid GMA embedments, offer a number of advantages over
other systems:
- Dehydration of tissues can be made directly in aqueous dilutions of GMA or optionally in
organic solvents
- GMA does not need to be water-free and indeed it works best with at least some water present.
- Infiltration of tissue with monomer can be performed at room temperature or lower.
- Polymerization of GMA can be performed at ambient temperature of 0°C with UV radiation
to 60°C in an oven.
- Thin sections of polymerized GMA can be cut with glass
or diamond knives.
- Sections from Low Acid GMA, unlike ordinary technical grade GMA, resist the uptake of stain,
thereby reducing greatly the occurrence of non-specific background staining.
- Enzyme digestion, a variety of stains and immunological localizations may be performed
on thick sections of GMA without removal of the plastic.
The Low Acid GMA Kit for TEM consists of three components, that is,
glycol methacrylate monomer (low acid), a polymerization initiator or catalyst (
benzoyl peroxide) and a plasticizer or filler (
butyl methacrylate). The GMA monomer is supplied with methacrylic acid substantially
removed and with small amounts of inhibitors present, to inhibit spontaneous polymerization
during synthesis and storage. This inhibitor need not be removed before use. The benzoyl
peroxide catalyst as supplied is provided in granular form.
Formulations:
The GMA embedding mixture for use with TEM applications is a modification of
the formula of Leduc1 (1967) which is used by
Cole2 (1981):
Recommended Amounts of GMA Components for TEM Applications
Component: Standard Formula for Final Medium
GMA (Low Acid): 67 ml
Water: 3 ml
Butyl Methacrylate: 30 ml
Benzoyl Peroxide: 0.60g
In the following preparative procedures, the 85% and 98% aqueous dilutions are made from the
above, as is the pre-polymerized GMA. The solubility of GMA in water may be increased by
reducing the temperature to 4-8°C. The complete, final medium, as shown above, can be
pre-polymerized.
Special comments about storage of the benzoyl peroxide: Avoid loss of water content from the
benzoyl peroxide as the dry material is considerable more hazardous and will burn vigorously.
Store below 38°C (100°F) to maintain product's activity.
Special safety comments about GMA use: The liquid GMA component may cause severe contact
dermatitis or allergic reactions in some individuals. Latex surgical gloves seem to help
prevent the contact dermatitis. The polymerized GMA block seems to be non-reactive and
non-toxic.
Mixing of the Resin Components:
The preparation and mixing of the resin components must be thorough and performed in a fume
hood. The mixture is stored in a brown glass bottle in a freezer and is stable for 6 months
or longer. Prior to use, allow the mixture to reach room temperature before opening the
container, to prevent condensation from contaminating the solution.
Special Precautions:
Avoid loss of water content from the benzoyl peroxide as the dry material is considerably
more hazardous and will burn vigorously. Store below 100°F (38°C) to maintain
activity.
The liquid GMA components may cause contact dermatitis or allergic reactions in some
individuals. Latex surgical gloves seem to help prevent the dermatitis. The polymerized
GMA appears non-toxic.
Dissolve the benzoyl peroxide in the water-glycol methacrylate-butyl methacrylate mixture.
Mix well and then pour enough of the solution into a 125ml or 250ml Erlenmeyer flask (Pyrex)
to form a shallow (5 to 7mm) layer. Heat slowly over a Bunsen burner with constant swirling.
The solution is polymerizing when the color deepens (to yellow-orange) and begins to thicken.
When the solution begins to polymerize, immediately plunge the flask into an ice water bath
and swirl vigorously until the solution is cooled to the temperature of the ice bath.
Polymerization is an exothermic reaction and once polymerization begins, rapid cooling is
imperative to prevent the very rapid complete polymerization in the flask. If the
proper viscosity is not reached after the initial heating and cooling, repeat the procedure
as many times as necessary to reach the desired end point. Properly pre-polymerized GMA
should have the viscosity of thick syrup at ice water temperature. That is, small air
bubbles within the solution should rise very slowly to the surface.
Dehydration, Infiltration and embedding
Small specimens (1mm3) or samples of tissue of a size
commonly used in TEM may be dehydrated in aqueous dilutions of glycol methacrylate.
Adequate tissue preservation is not maintained if large samples are dehydrated with GMA.
After rinsing of the fixed* tissue in buffer or water, specimens or tissue samples are
transferred directly to 80% GMA in water. A typical schedule for GMA dehydration, infiltration
and embedding is as follows:
Rinse 5 min. to 1 hr.
85% GMA 2 changes, 1 hr. ea.
97% GMA 2 changes, 1 hr. ea.
Final GMA
embedding mixture Overnight (not pre-polymerized)
For final embedding:
Place the infiltrated tissue and a drop or two of pre-polymerized GMA in a
Size 00 gelatin capsule, orient
the tissue as desired, insert identification tag, fill the capsules to the top or
to form a positive meniscus with pre-polymerized GMA, and cap. Oxygen inhibits polymerization.
Place the capsules upright in the capsule holders and place in
ultraviolet apparatus equipped with 2 long wavelength (3150A) UV lamps (G.E. #F6T5,BL) until
polymerized (12-24 hours). In the presence of air or higher water content, 2-3 days may be
required to complete polymerization.
*Fixing with OsO4 should be avoided. See ref. #4.
The plastic may be considered polymerized when the upper surface of the plastic appears
whitish, or when the surface cannot be dented with your thumbnail.
The gelatin capsule may be peeled off after it has been softened in water. Wait at least
24 hours to section if gelatin was removed after soaking in water. GMA is humidity sensitive.
If blocks are too hard to section, place them in a humid atmosphere for 1-2 hrs. Conversely,
if the blocks are too soft, place them in a desiccator for 1-2 hrs. Polymerized blocks may be
stored at room temperature indefinitely.
Thin Sectioning:
When thin sectioning, the GMA cutting characteristics differ considerably from epoxy type,
non-water soluble resins. Best results are obtained when the water level in the knife boat
is kept to a minimum; just high enough to wet the knife edge. It has also been observed that
GMA cuts better when the relative humidity is 50% or less. GMA also sections easier at higher
cutting speeds and a more acute knife angle than epoxy resins.
If combined LM and TEM studies are to be carried out employing the same embeddment, it is
recommended that the TEM formulation be used, since it is easier to cut thick sections from
the TEM blocks than to cut thin sections from the blocks prepared for LM.
References:
1Leduc, E.H. and Bernhard, W., J. Ultrastruct. Res. 19, 196 (1967).
2Cole, M.B., Jr. Personal Communication, 1981.
3Cole, M.B., Jr. J. Microscopie (Paris) 7, 441 (1968)
4Barsotti, P. et al. J. Submicrosc. Cytol. 12, 233 (1980)
Disclaimer
The information given in this bulletin is to the best of our knowledge accurate, but no
warranty is expressed or implied. It is the user's responsibility to determine the suitability
for his own use of the products described herein; and since conditions of use are beyond our
control, we disclaim all liability with respect to the use of any material supplied by us.
Nothing contained herein shall be construed as a recommendation to practice any patented
invention nor as a recommendation to use any product or to practice any process in violation
of any law or any government regulation.
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Sunday March 21, 2010
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