LR Gold™ Resin Systems

Technical Data Sheet: Processing and Polymerization

While tissue samples up to 5 x 5 x 5 mm have been successfully processed using the following schedule, it is recommended that tissue specimens no longer than 3 x 3 x 3 mm be used. Further, as a general rule the smaller the specimen the more efficient the impregnation. The thickness of the tissue is particularly important when polymerizing darkly colored tissue such as liver and spleen, because complete polymerization depends on the blue light from the light source being able to penetrate the full thickness of the tissue. Tissue used should be fresh and unfixed.

Processing

Processing is performed in 10 ml vials with tight fitting lids on a rotary agitator at the temperatures listed in the table below. The fluids involved are stored in bulk at the sub-zero temperatures required. Also, it must be kept in mind, the mixed resin are sensitive to prolonged light exposure and are therefore stored in the dark and handled as infrequently as possible. We recommend the use of polyvinyl pyrrolidone to protect unfixed tissue from osmotic changes during processing. We have used PVP with an approximate molecular weight of 44,000. This can be dissolved in methanol, water, or the London Resin Gold monomer. Concentrations of 50% w/v are possible in the methanol mixtures, however, at low temperatures, the higher viscosity is impractical. The following schedule shows the recommended PVP concentrations.

While this protocol yields very satisfactory results for LM work, it must be said, that the addition of PVP in different concentrations may further improve morphology, especially in the EM.

Fresh Tissue

50% methanol, 20% in PVP*	0°C	15 min.
50% methanol, 10% in PVP	-25°C	45 min.
30% methanol, 10% in PVP	-25°C	45 min.
50% LRGold monomer / 50% LRGold methanol, 10% in PVP	-25°C	30 min.
70% LRGold monomer / 30% LRGold methanol, 10% in PVP	-25°C	60 min.
100% LRGold monomer	-25°C	60 min.
100% LRGold monomer + initiator	-25°C	60 min.
100% LRGold monomer + initiator	-25°C	Overnight
100% LRGold monomer + initiator	-25°C	20-25 hours polymerization
* 20g PVP in 100ml methanol

Polymerization

The addition of a light sensitive initiator is needed in order to polymerize the resin and we recommend Benzil, an alpha-diketone, at a concentration of 0.1% w/v. The principle is shown in the diagram below.

It is not advisable to mount sections onto slides before
reacting, since this involves heat and would be deleterious
to the unfixed proteins. The section can of course be
mounted in the usual way after enzyme histochemistry
or immunocytochemistry has been carried out.

The gelatin capsules are 00 size and the plastic support is a modified hemagglutination tray. The bulb involved here is a Thorn projector lamp (AI/209 FDX, 12V 100W). We have found that 7 to 9 V will induce solidification within 24 hours. As with many acrylic resins oxygen will inhibit polymerization, therefore, the capsules should be filled completely and lids fitted. Paper labels can be inserted into the capsules. Nine capsules (3 x 3) may be polymerized at anyone time. If the upper surface is still soft after 24 hours, it can be trimmed off or hardened in daylight for a few hours prior to peeling off the gelatin. Once polymerized, the blocks need not be stored cold, but storage in the cold may prolong the activity of some enzymes.

Enzyme histochemistry performed to date using this resin has involved conventional reagents, times of reaction and temperatures (see Thompson and Germain, Histochemical Journal Vol. 15, No. 12, December 1983).

For room temperature polymerization, using a peroxide/amine cure, add to pure LRGold either 1% of dry benzoyl peroxide or, more safely, 1.5% of benzoyl peroxide paste (60% in dibutyl phthalate). Infiltrate with pure LRGold solution, adding the peroxide mix just prior to polymerization. To reduce the heat of curing, cool the mold in ice water. To accelerate the cure, add 1 drop of LRWhite accelerator to 20ml LRGold resin/benzoyl peroxide mixture. To U.V. light cure, add benzoin methyl ether. The precise concentration of the ether will depend on the power and emission spectrum of your UV lamp. However, a useful starting concentration would be 0.5%.

The cross link density of the final resin is important. If stains are not penetrating sufficiently quickly, reduce the benzil concentration rather than the light intensity or exposure time.

The LRGold blocks, following polymerization, can be stored, handled and cut at room temperature. Cutting should preferably be done using a motorized microtome and a glass knife. Sections may be cut dry, picked up and placed free-floating into incubating medium or buffer wash for enzyme histochemistry or immunocytochemistry.

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