GMA (Glycol methacrylate) embedding for immmunohistochemistry protocol

Advantages and procedure for GMA embedding of small biopsy samples.

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Advantages of using GMA

Water miscible, doesn’t require dehydration and rehydration steps
No need to eliminate resin before staining
Low viscosity, penetrates tissue easily
No crosslinking, no antigen retrieval
Good antigen presentation
Good morphology preservation (cellular localisation)
Low temperature processing
Can cut very thin sections (1-2 μm) making the most of very small biopsies – very good resolution

Fixation

Several methods of tissue fixation can be used for GMA. Fixing in acetone usually gives good results For example:

  1. Place biopsy immediately in ice cold acetone containing protease inhibitors
  2. Fix overnight -20°C
  3. Replace fixative with acetone (room temperature) 15 min

Processing

  1. Place biopsy in Methyl benzoate for 15 minutes (This helps infiltration of GMA into the tissue)
  2. Place biopsy in 5% methyl benzoate in GMA 4°C. Three times for two hours

Embedding

Follow the kit manufacturer’s instructions for embedding into GMA itself. The GMA will need to be polymerised using a catalyst (provided in commercially available kits) and left to set for 48 hours at 4°C

Section preparation

Sections can be cut at 1-2 μm.

Lay sections out on a water bath containing ammonia (2 ml ammonia in 1 L distilled water). No need to heat (as with paraffin sections). The ammonia helps antigenicity and provides better antibody staining (although the mechanisms for this are not clear).

Sections can be picked up on 10% poly-l-lysine coated slides and dried ready for staining. (wrap in foil and store at -20°C for no longer than two weeks)