Glycol Methacrylate Embedding for Soft Tissues
Technovit Glycol Methacrylate: Technical Information and Help From Heraeus Kulzer.
Embedding in plastic provides many advantages to the histotechnologist. Thinner sections can be made providing improved detail. Better support is given to cellular components, offering improved morphology. Short, straight-forward protocols are available giving minimum processing times. Improved chemistry provided by kits gives uniform results and a wider range of application.
While this protocol will help get you started with embedding your soft biological samples in the Technovit GMA kits sold by Energy Beam Sciences, it follows the general guidelines for embedding in any source of GMA.
Fixation of the biological samples can be done in any way appropriate for the work you are doing. Immersion or perfusion with 4% neutralized formaldehyde, prepared from paraformaldehyde according to Karnovsky, is usually preferred. Try to keep the sample size small using 10mm x 10mm x 2mm as a maximum.
Dehydration for GMA embedments does not have to be complete because of GMA's miscibility in water. Use a schedule of increasing alcohol concentration at room temperature as follows:
- 70% ethanol for 2 hours.
- 96% ethanol for 2 hours.
- 96% ethanol for 2 hours.
- absolute ethanol for 1 hour.
A short defatting step can help with infiltration. If desired, submerge the sample in acetone for 10 minutes.
Make up your infiltrating solution from:
- 100 ml of Technovit 7100 resin, i.e. 2-hydroxyethyl methacrylate (GMA)
- 1 gm of Hardener I (benzoyl peroxide)
Mix using a magnetic stirrer until the benzoyl peroxide is completely dissolved. Store at 4°C in a dark bottle for up to 2 months.
Infiltrate in a 50/50 mixture of 100% ethanol and infiltration solution for two hours. Leave the sample in 100% infiltration solution overnight. Infiltration with mild agitation and/or vacuum will be more complete and larger samples should have more infiltration steps over a longer period of time.
Make up your embedding solution from:
- 15 parts of infiltrating solution
- 1 part of Hardener II
Mix for 1 minute using a magnetic stirrer. Use the solution within 10 minutes, before polymerization occurs.
Cover your samples in the embedding molds with this solution. Molds of polyethylene such as the JB-4 molds or Peel-Away molds are most often used. Block holder/adapter in the recess on the mold and add mounting plastic. Begin sectioning with glass.
Additional Online Resources
Technovit Glycol Methacrylate Embedding Kits
Plastic Embedded Tissue: More Techniques and Protocols
Light Microscopy Supplies