Rapid embedding methods into epoxy and LR White resins for morphological and immunological analysis of cryofixed biological specimens

Microsc Microanal. 2014 Feb;20(1):152-63. doi: 10.1017/S1431927613013846. Epub 2013 Nov 19.

Abstract

A variety of specimens including bacteria, ciliates, choanoflagellates (Salpingoeca rosetta), zebrafish (Danio rerio) embryos, nematode worms (Caenorhabditis elegans), and leaves of white clover (Trifolium repens) plants were high pressure frozen, freeze-substituted, infiltrated with either Epon, Epon-Araldite, or LR White resins, and polymerized. Total processing time from freezing to blocks ready to section was about 6 h. For epoxy embedding the specimens were freeze-substituted in 1% osmium tetroxide plus 0.1% uranyl acetate in acetone. For embedding in LR White the freeze-substitution medium was 0.2% uranyl acetate in acetone. Rapid infiltration was achieved by centrifugation through increasing concentrations of resin followed by polymerization at 100°C for 1.5-2 h. The preservation of ultrastructure was comparable to standard freeze substitution and resin embedding methods that take days to complete. On-section immunolabeling results for actin and tubulin molecules were positive with very low background labeling. The LR White methods offer a safer, quicker, and less-expensive alternative to Lowicryl embedding of specimens processed for on-section immunolabeling without traditional aldehyde fixatives.

MeSH terms

  • Animals
  • Bacteria
  • Epoxy Resins
  • Freeze Substitution / methods*
  • Immunohistochemistry / methods*
  • Plant Leaves
  • Tissue Embedding / methods*

Substances

  • Epoxy Resins