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Preservation of vascular glycocalyx ultrastructure
Author(s) -
Sims David Edward,
Horne Margaret
Publication year - 2008
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.22.2_supplement.36
Subject(s) - fixative , glutaraldehyde , osmium tetroxide , glycocalyx , aqueous solution , chemistry , chromatography , osmium , fixation (population genetics) , biophysics , electron microscope , biochemistry , organic chemistry , biology , physics , cytoplasm , ruthenium , optics , gene , catalysis
Preservation of vascular glycocalyx has proven problematic. Complex carbohydrates that constitute most of glycocalyx are disrupted by physiological stresses and aqueous fixatives. This study found that a non‐aqueous, osmium‐based fixative was superior to aqueous alternatives. Buffered aqueous fixative (2.5% glutaraldehyde in 0.05M sodium cacodylate, pH=7.3) was compared with a non‐aqueous fixative composed of 0.05% osmium tetroxide dissolved in fluorocarbon (FC‐72, 3M Co.). Samples of perfused rat aortas were processed for ultrastructural analysis immediately after initial fixation, or after various periods of time in non‐aqueous solvent, non‐aqueous fixative, buffered glutaraldehyde or aqueous buffer. Glycocalyx composed of mildly electron‐dense branching materials up to 600 nm tall was best preserved by non‐aqueous fixation followed by immediate processing. Post‐fixation delays for up to 24 hours in either non‐aqueous solvent or the solvent mixed with osmium did not substantially alter its appearance. However, the same samples, if post‐fixation‐treated with buffer solution or buffered glutaraldehyde for 30 minutes had no appreciable glycocalyx remaining. Non‐aqueous fixation offered the further advantage of exerting no osmotic forces, so there was no appreciable shrinking of cells or separation from connective tissues.