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Electron Microscopy Sciences

Technical Data Sheets

Vestopal-W Embedding Medium

EMS Catalog #14700

Vestopal is a copolymer between a polyester of maleic and fumaric acids, esterified with di- and tri- hydroxyalcohols and styrene. In Vestopal, as supplied, styrene is mainly present as the monomer although some copolymer with the polyester is usually present. Vestopal "monomer" may be stored at -15°C. For several months without noticeable change.

Because this plastic is not miscible with water, biological specimens must be thoroughly dehydrated before infiltration with Vestopal. Dehydration may be carried out with acetone, dioxane, or glycol methacrylate.

Procedure for Vestopal

The following procedure illustrates a typical use of Vestopal.

  1. Fixed material is dehydrated by successive passage through 25, 50, 75 and 90% solutions of acetone in water, 20 minutes in each, and then by immersion in two changes of 100% acetone for at least 30 minutes each.
  2. The dehydrated material is then immersed for at least 30 minutes in each of four different Vestopal mixtures. Mixture I contains 1 part Vestopal plus 3 parts acetone; mixture II contains 1 part Vestopal plus 1 part acetone; mixture III contains 3 parts Vestopal plus 1 part acetone; and mixture IV contains 99% Vestopal, 0.5% tertiary butyl perbenzoate, and 0.5% of 3% solution of cobalt naphthenate in styrene. (If harder blocks are desired, increase the concentration of t-butyl perbenzoate to 1%.)
  3. This infiltration material is usually passed through at least one additional change of mixture IV (it may be kept in mixture IV overnight, provided the room is not so warm as to cause a significant degree of polymerization).
  4. The specimens are then places in gelatin capsules filled with mixture IV. Polymerization is achieved by keeping the filled capsules at 60°C. For 12 - 24 hours.

The hardness of the blocks, as already mentioned, is controlled by the amount of t-butyl perbenzoate in the final embedding mixture. It is also possible to produce softer blocks by the addition of dibutyl phthalate.

It is possible to polymerize Vestopal with ultraviolet light. Cobalt naphthenate may be omitted from mixture IV if UV polymerization is desired. Depending on the UV source, polymerization usually takes 2 - 4 hours.

One additional modification of the above procedure may be of interest: Rather than transferring specimens through several Vestopal-acetone mixtures (step 2), the specimen may be placed in a solution of 25% Vestopal in Acetone. The acetone is then allowed to gradually evaporate (usually overnight). This procedure results in complete infiltration of the specimen. Care should be taken, however, that moisture does not condense on the plastic as it is cooled by the evaporating acetone. The plastic will acquire a slightly white appearance should water vapor condense on it.

Cobalt naphthenate included in our kit is a 6% solution. Immediately prior to use, it should be diluted to a 3% solution by mixing with an equal volume of styrene.

DO NOT MIX COBALT NAPHTHENATE AND t-BUTYL PERBENZOATE DIRECTLY TOGETHER, AS THIS COMBINATION MAY BE EXPLOSIVE. ALWAYS ADD THESE REAGENTS TO THE VESTOPAL MIXTURE.

References

Kellenberger, Ryter, and Sechaud, J. Biophys., Biochem., Cytol 4:671, (1958).

Ryter, A. And Kellenberger, E., J. Ultrastruct. Res. 2:200 (1958)

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Vestopal-W Embedding Medium