Embedding Protocols for Bone Histology
Welcome! Below you’ll find detailed information on how to prepare archaeological bone samples for embedding in epoxy resin, low-viscosity epoxy resin, polyester resin and methyl methacrylate. This information is supplemental to our team’s poster presentation at the 2022 United Kingdom Archaeological Sciences (UKAS) Conference at the University of Aberdeen. Protocols below include lessons learned from our study. Deviations from our study methods are numbered in text and detailed at the bottom of the page.
ALWAYS READ SAFETY DATA SHEETS PRIOR TO USE. FOLLOW ALL INSTRUCTIONS ON EMBEDDING CHEMICALS.
UKAS, Poster Session April 21, 2022
Preparing undecalcified bone samples for embedding involves:
Dehydrate to ensure fat and water content removed from bone
Infiltrate sample with increasing concentration of resin
Cure/Polymerise in embedding moulds
Generally, cremated or otherwise collagen-poor archaeological bone is already dehydrated and the first step is skipped. Fresh bone requires treatment with Tergazyme, xylene and/or acetone prior to infiltration.
Image: Histological section of calcined bone from an early medieval cremation
Epoxy.
Infiltration Schedule:
Day 1: 100% ethanol (cover)
Day 2: 25% resin, 75% ethanol (cover)
Day 3: 50% resin, ethanol (cover)
Day 4: 75% resin, 25% ethanol (cover)
Day 5-6: 100% resin changed every 24 hours
Curing Protocol: Stir resin and hardener (100 grams resin to 12 grams hardener) for at least 2 minutes. Pour over samples in embedding moulds. Place in vacuum chamber with vacuum pump to remove all air bubbles (1). Samples cure at ambient temperature in 12 hours.
Low Viscosity Epoxy.
Infiltration Schedule:
Day 1: 100% ethanol (cover)
Day 2: Mix resin, hardener, and accelerant in resin bottle and store in 4 C fridge. Infiltrate with 25% resin mix, 75% ethanol (cover)
Day 3: 50% resin mix, ethanol (cover)
Day 4: 75% resin mix, 25% ethanol (cover)
Day 5-6: 100% resin mix changed every 24 hours
Curing Protocol: Pour resin mix over samples in embedding moulds. Place in vacuum chamber with vacuum pump to remove all air bubbles (1). Place samples in oven set to 70 C (2). Samples cure in 72 hours.
Polyester.
Infiltration Schedule: Resin is stored at 4 C.
Day 1: 100% ethanol (cover)
Day 2: 25% resin, 75% ethanol (cover)
Day 3: 50% resin, ethanol (cover)
Day 4: 75% resin, 25% ethanol (cover)
Day 5-6: 100% resin changed every 24 hours
Curing Protocol: Mix resin and methyl ethyl ketone peroxide (MEXP) catalyst (0.1% total volume) and stir well. Pour over samples in embedding moulds. Place in vacuum chamber with vacuum pump to remove all air bubbles (1). Place samples in oven set to 70 C (2). Samples cure in 72 hours.
MMA.
Infiltration Schedule: MMA (with MEHQ as inhibitor) is stored at 4 C.
Day 1: 100% ethanol (cover)
Day 2: 25% resin mix, 75% ethanol in covered beaker. Place in rotary mixer.
Day 3: 50% resin mix, ethanol in covered beaker. Place in rotary mixer.
Day 4: 75% resin mix, 25% ethanol in covered beaker. Place in rotary mixer.
Day 5-6: 100% resin changed every 24 hours into covered beaker. Continue using rotary mixer.
Polymerisation Protocol: A photo synthesizer (Benzoin ethyl ether) is added to MMA at 0.3% total volume. The resin mixture is degassed with zero-grade nitrogen gas for about 3 minutes. Pour over samples in embedding moulds. Store unused resin at -20 C. Place samples in vacuum chamber with vacuum pump to remove all air bubbles (1). Place samples in Reichert/Leica Automatic Freeze Substitution (AFS) chamber lined with aluminum foil. The AFS machine provides an oxygen-depleted/ nitrogen-rich environment, is fitted with UV lights and holds samples at constant -25 C temperature. Samples polymerise in 96 hours (3).
Original protocol did not utilise a vacuum pump during the infiltration stage or just prior to embedding. Final embedded blocks either had large voids (esp true of MMA blocks), bubbles in trabeculae or voids in midcortical microcracks. One hypothesis is that the low viscosity epoxy resin may have performed the best due to its ability to impregnate these areas.
Our oven was set to 50 C and some of the low viscosity epoxy and polyester blocks were tacky, especially in mid-block around the sample. We recommend increasing cure temperature to 70 C.
Our samples were in Reichert-AFS to polymerise under UV light for 48 hours. This is sufficient for smaller biological samples usually processed in the OPTOM (Cardiff) laboratory but was insufficient for the volume in our 22 mm x 22 mm x 20 mm moulds. We recommend polymerising under UV lights for at least 96 hours to ensure adequate polymerisation.