GMA Embedding Protocol

GMA stands for "Glycol Methacrylate," and GMA embedding is a histological technique used for the preparation of tissue samples for microscopic analysis. GMA embedding involves infiltrating and embedding biological tissue specimens in a polymerized GMA resin, allowing for the production of thin sections for histological examination under a microscope. This method is particularly useful for preserving tissue morphology and enhancing the quality of tissue sections for various histological and immunohistochemical analyses.

Fixation

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

Processing

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

Embedding

• Follow the instructions for embedding into GMA itself. The GMA will need to be polymerized 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).

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• Before GMA infiltration, the tissue specimens must undergo thorough dehydration to remove water content. Gradual ethanol series or other dehydration agents are typically employed to aid in this process, and the selection of dehydration protocol should be optimized based on the tissue type and size.
• The GMA resin should be prepared and infiltrated under controlled conditions to optimize the penetration of the resin into the tissue. This includes the use of appropriate ratios and concentrations of GMA monomer, along with the consideration of temperature and duration of infiltration to ensure complete impregnation of the tissue.
• Suitable embedding molds of the desired size and shape should be selected to accommodate the tissue and the subsequent sectioning. Proper orientation of the tissue samples within the molds is essential to facilitate accurate sectioning and subsequent microscopic analysis.
• The polymerization process should be carefully monitored to ensure complete and uniform solidification of the GMA resin. This may involve the use of specific initiators and optimized temperature and time parameters, along with protection from light or other factors that may interfere with the polymerization reaction.

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