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A water - miscible embedding media is ideal for the preparation of tissues for cyto-chemical studies and enzyme localization when a correlation between light microscopy and electron microscopy is necessary, i.e., to examine a thick and a thin sections from the same block.
Embedding techniques have been improved by:
Two embedding mixtures are recommended by SPAUR and MORIARTY (1977).
Glycol Methacrylate 95% GMA (7 parts) | 66.5 ml. |
Distilled Water | 3.5 ml. |
n-Butyl Methacrylate (3 parts) | 28.5 ml. |
Ethylene Dimethacrylate (5% v/v) | 5.0 ml. |
Benzoyl Peroxide (1.5% w/v) | 1.5 gm. |
Glycol Methacrylate 100% GMA (7 parts) | 66.5 ml. |
n-Butyl Methacrylate (3 parts) | 28.5 ml. |
Ethylene Dimethacrylate (5% v/v) | 5.0 ml. |
Benzoyl Peroxide (1.5% w/v) | 1.5 gm. |
Polyethylene Glycol 400 (1% v/v) | 1.0 ml. |
The following materials are needed: a narrow-mouth, 125 ml Erlenmeyer flask; a #5 stopper with two holes, one at an angle to accommodate a thermometer; a short-bulb thermometer; a magnetic-stirrer hot-plate; and a dry ice-ethanol bath in 1000 ml. beaker.
* The 98°C. temperature is only suggested. It may vary 1 - 2°C. less if the polymerization reaction is strong, and one degree less will give the prepolymer a good viscosity, i.e., a thick syrup at 0 - 4°C.
Dehydration and infiltration can be done at room temperature, and also can be done at 4°C. on ice, with the time extended for each step.
Modified Dehydration Schedule - complete with glycol methacrylate monomer - GMA | |
80% GMA in distilled water | 2 changes 10 minutes each |
95% GMA in distilled water | 10 minutes |
100% GMA | 3 changes 10 minutes each |
Anhydrous unprepolymerized mixture | 15 minutes |
Prepolymerized mixture | 24 hours |
Dilutions of GMA are made with distilled water. The anhydrous mixture is made in the same proportions of the embedding mixture except without water and 100% GMA. (See Mixture I.) This procedure ensures a complete dehydration and a good infiltration with results comparable to those achieved using Araldite 6005.
The tissue is embedding in gelatin capsules (not polyethylene) with a fresh prepolymer mixture. Leave the capsules open for 30 minutes to eliminate air bubbles, or for 10 minutes in a vacuum chamber, then cap with as little air as possible trapped within the capsule. The polymerization is accomplished by placing the capsule 10 - 20 mm under a long wavelength, ultraviolet light source (3150 W) for 12 to 16 hours, depending on the block, it can be accomplished by irradiation of the top for 1 - 2 hours. Bubble formation is a occurrence and causes no normal problems.
The gelatin capsules can be removed after polymerization by soaking in warm water until all gelatin is dissolved.
Sections can be collected on coated or bare copper grids (200 mesh), and stained by routine methods with Uranyl acetate and Lead citrate.
Glycol methacrylate (GMA) is a useful embedding medium for sections for High Resolution Light Microscopy. Sections of 1 to 1 micron can be cut with a conventional rotary microtome and steel knife, or glass Ralph Knives, and stained with a variety of special stains. This technique is of special use for biopsies when a light and electron microscope are needed.
PEG - GMA Embedding Medium
Glycol Methacrylate, GMA (Low Acid) MSDS
n-Butyl Methacrylate MSDS
Ethylene Glycol Dimethacrylate MSDS
Polyethylene Glycol 400 MSDS
Benzoyl Peroxide Paste MSDS