Introduction
Araldite 502 is an epoxy resin used as an embedding medium for electron microscopy. It yields a light gold colour, hard block. Tissues to be embedded in Araldite 502 can be dehydrated with ethanol or acetone. These solvents must be pure and after the third absolute ethanol/acetone change, prior to mixing with liquid resin, a transitional solvent Propylene Oxide (PO), was originally recommended. PO is expensive and is a recognized carcinogen. If you wish to avoid using PO, then make an additional change using pure ethanol or acetone which has be stored with dried Molecular Sieve 4A in a well sealed jar. Use roughly 10% by volume, then the molecular sieve will further reduce water within organic solvents and the resulting blocks will not be soft or sticky due to water contamination. Tissues to be embedded in Araldite 502 can be dehydrated with most commonly used organic solvents. However the application of a transitional solvent, such as Propylene Oxide, is advisable because epoxy resins are more soluble in propylene oxide.
Recommended Procedure
Fixation
Tissues can be fixed in a wide range of fixatives. One of the more commonly used fixatives is an Aldehyde (i.e.: Glutaraldehyde) followed by Osmium Tetroxide.
Dehydration
There are many different dehydration schedules that can be followed. A typical one is as follows:
- 70% Ethanol for 5 minutes
- 95% Ethanol for 5 minutes
- 100% Ethanol for 5 minutes
- 100% Ethanol for 15 minutes
- 100% Propylene Oxide for 5 minutes
- 100% Propylene Oxide for 15 minutes
- Note: Longer times may be required for some samples. Times are fairly arbitrary, but compounding short-cuts may cause problems. Sensitive specimen (fresh water algae) would be processed using very weak buffers and starting dehydration no higher than at 30% and applying more, smaller increases in solvent concentration.
Ingredients
- Araldite 502 - 20mL
- Dodecenyl Succinic Anhydride (DDSA) - 22mL
- Benzyldimethylamine (BDMA) - 1-1.2mL
- BDMA is recommended for better penetration and stability, however you could also use
- 2,2-Dimethoxypropane (DMP) - 0.63-0.84mL
- For larger batches simply increase each component proportionally.
- ProSciTech Pty Ltd offers an Araldite Embedding Kit as a cheaper alternative to buying each of these chemicals separately.
Mixing Instructions
Slight variations of the accelerator (DMP-30 or BDMA) will drastically affect the colour and brittleness of the block.
Prior to measuring and mixing, the resin and the anhydride should be warmed (60°C) to reduce their viscosity. Thorough mixing is imperative to be able to achieve uniform blocks. The final block can be made harder by replacing some of the DDSA with Methyl-5-norbornene-2,3-dicarboxylic anhydride (NMA) (0.5mL of NMA for each 1.0mL of DDSA).
Although the mixture can be stored for up to 6 months at 4°C, it is highly recommended that freshly prepared embedding medium always be used. If you choose to store the mixture, you should warm it thoroughly prior to adding the accelerator.
Infiltration
It is recommended that for all of the infiltration steps a specimen rotator be used.
- Drain the tissue of most of the propylene oxide, leaving a little so the tissue does not dry out.
- Replace the solvent with a 1:1 solution of propylene oxide:embedding medium and allow it to stand for at least 1 hour at room temperature.
- Remove the mixture, replace it with 100% embedding medium and leave for 6-12 hours at room temperature.
Embedding
Transfer each sample to a dry capsule or mould and fill the mould with embedding medium. Cure the medium in an oven at 60°C for 12 hours or until it is hard. Block hardness increases with curing time and temperature. If a curing oven is plugged into a lamp lighter/timer, this may be set to run at night and blocks will always receive the same curing time if placed into the oven during day hours and if they are removed any time on the following day. Araldite is very viscous and infiltrates poorly, but it stains and sections better than Spurr's resin.
Blocks can be trimmed and sectioned after the blocks return to room temperature.
References
- Finck, H. (1960), J. Biophys. Biochem. Cytol. 7, 27-30
- Luft, J.H.(1961), J. Biophys. Biochem. Cytol. 9, 409-414
- Glauert, A.(1991), Microscopy and Analysis, September; 15-20