Fixation on Histology

Epoxy resins are used in conventional transmission electron microscopy and high-resolution light microscopy applications such as osmium-paraphenylenediamine staining of myelinated axons in neuronal tissue on 1-um semithin sections.  The ‘pot-life’ of most epoxy-based resins (e.g. EPON812 and Spurr’s low viscosity) are within a few days after mixture of the accelerator according to vendor formulations and instructions. Epoxy mixture formulations may be scaled down yet residual amounts of resin may be remaining in the ‘pot’.  Given the high cost of these resin components and kits every milliliter drop should be used especially in research labs that receive samples with varying sample numbers at different times. Having a pre-mixed stock can save costs, time resources in preparation and disposal clean up.

Remaining fresh epoxy resin from the ‘pot’ may be stored frozen in disposable polypropylene syringes that decrease the polymerization viscosity increase, prevent air-moisture exposure and able to have pre-mixed stock on hand for processing. These frozen aliquots of epoxy resins (e.g EPON812 and Spurr’s low viscosity) are routinely used in our lab for semithin histology and TEM processing tissue samples for the initial infiltration solutions. Epoxy resin mixtures are prepared according to vendor instructions (e.g Tousimis, cat.# 3132, tEPON 812 epoxy resin kit,  medium formulation) measured out in using graded volume cylinders and mixed in 50 ml polypropylene disposable beakers,, manually stirred with a flat wooden spatula and then  with a magnetic stirrer for a few minutes.  The mixtures are used in an automated EMS Lynx II EM tissue processor (Electron Microscopy Sciences, Hatfield, PA) for infiltration solutions of 1:4, 1:2, 3:1, Epon812 resin: Propylene oxide, then neat Epon812 resin using agitation and vacuum.  We do not use frozen stock epoxy resin for neat resin infiltration or final embedding and dispose all infiltration solutions after use.  In addition, we do not re-use neat infiltration epoxy resin after use in the processor due to the solvent contamination and polymerization and viscosity increase. 

Within the freshly prepared ‘pot’, the residual amounts of epoxy resin may be suctioned up into polypropylene syringes (1 ml, 6 mL, 10ml, or 12 mL) then elevated vertically to lift the air bubbles to the top.  After expulsion of air bubbles from syringe, the dispensing tip is sealed with Parafilm^TM, labelled with contents and date, wrapped in aluminum foil then placed in a sealed plastic bag and stored in a -20^oC freezer for a period of days to up to 4 weeks, maximum, until use.  When we use the frozen stock aliquots of EPON 812 resin loaded in the syringes, they are thawed up to room temperature within 1-hour then mixed with propylene oxide as 1:4 and 1:2 EPON812:propylene oxide infiltration solutions for use in the EM tissue processor.  Another use of the frozen stock can be for use in adhesive mounting and re-orienting sample blocks which occasionally occurs due to various factors of the processed sample falling during polymerization within the mold or sample needs to be sectioned at a different plane to reach the region-of-interest.  If the frozen stock becomes highly viscous then one should reconsider using as an infiltration solution.  Our laboratory routinely processes over 500 semithin and TEM samples annually using frozen stock epoxy resin for infiltration solutions and has not had processing related issues and the polymerized blocks are identical in infiltration and hardness whether frozen stock or freshly prepared epoxy resin was used in the infiltration solutions during processing.

 

For more information on resin processing there are excellent resource guides available to all NSH members. Not an NSH Member? Join now to gain access.

• Hard Tissue Troubleshooting Guide on Tissue Processing Schedules and Embedding Methods for Hard Tissue
• Troubleshooting Resources (Search for ‘Hard Tissue’ for additional resource guides).

Written By: Philip Seifert, HTL(ASCP)