Glycol Methacrylate Embedding for Immunohistochemistry
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. In addition, improved chemistry and protocols
are now available that are more antigen-friendly.
This protocol will help get you started with embedding your soft
biological samples in Technovit 8100, a GMA kit sold by Energy Beam Sciences.
Note the areas in which it deviates from the guidelines for embedding
in GMA for morphology only.
Use gentle agitation during fixation, washing, dehydration
and infiltration for best results.
Fixation of the biological samples can be
done in any way appropriate for the antigen you are seeking.
Cold acetone, periodate/lysine/paraformaldehyde,
and buffered paraformaldehyde solutions have all been used successfully.
Try to keep the sample size small using 10mm x 10mm x 2 mm as a
maximum. Fixation should be done for several hours at 4°C.
Wash out the fixative with 6% sucrose solution
at 4°C overnight.
Dehydration for GMA embedments does not have
to be complete because of GMA's miscibility in water. Use acetone
for 1 hour at 4° C changing
the acetone at the beginning until it remains clear.
Make up your infiltrating solution from:
- 100 ml of Technovit 8100 resin, i.e. 2-hydroxyethyl methacrylate
- 0.6 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 1 month.
Infiltrate for 6 - 10 hours at 4°C using mild
agitation. Vacuum will make infiltration more complete and larger
have more infiltration steps over a longer period of time.
Make up your embedding solution from:
- 30 ml of infiltrating solution
- 1 ml 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 or if
needed, prepare silanized slides.