Chemical fixation and acrylic resin embedding
This technique combines gentle chemical fixation with embedding in a hydrophilic acrylic resin such as LR Gold.
Osmium postfixation is typically avoided. In a special variation of this technique called progressive lowering of temperature (PLT) dehydrating solvent and resin infiltration are performed at sub-zero temperatures in a freeze substitution machine. The temperature is gradually lowered from 4°C to ‑25°C to minimise extraction of cell content by the solvent and resin. The resin can be polymerised using UV light instead of heat to prevent any further loss of antigenicity. PLT is appropriate for certain proteins while the room temperature variations of the protocol work well for many plant cell wall carbohydrates.
Pectic homogoalacturonan (carbohydrate epitope) labeling in haustorial cell walls and paramural deposits of Rhinanthus minor. In this preparation, even the osmication step was included without detrimental effect on labelling. Scalebar: 500 nm. Preparation and image: Anna Pielach.