Variations in fixation techniques for field emission SEM and TEM of zebrafish (Branchydanio rerio) embryo inner and outer membranes

The morphology of embryos of the fresh water teleost, Brachydania rerio (ze brafish), was examined in a parallel FE-SEM/TEM study, after various pre- a nd post-fixation regimes. Special attention was paid to the chorion, the co ntents of the peri-vitelline space, the plasma membrane, the syncytial laye r, the blastodermal cells and the yolk. The preservation of these rather di fferent structures with their varied chemical constituents was not straight forward. Standard fixation GA + OsO4 (glutaraldehyde, osmium tetroxide) did not adequately preserve these structures, and although well established no n-coating techniques gave, with GA as prefixative, an acceptable image in F E-SEMI the preservation for TEM was inferior. The opposite result was obtai ned with a combination of tannic acid, OsO4 and uranyl acetate. Using this mixture the results with FE-SEM were inferior, while the TEM results were m uch improved. Introduction of a mixture of GA, PF (paraformaldehyde), acrol ein and picric acid as prefixative, in combination with the TAO (tannic aci d/arginine/OsO4) non-coating post-fixation technique for FE-SEM and the OsO 4/K4Fe(CN)(6) (osmium tetroxide/potassium ferrocyanide) post-fixation metho d for TEM? respectively, resulted in an adequate preservation of the chorio n, plasma membrane, syncytial layer and cells of the blastoderm.