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 (zebrafish), was examined in a parallel FE-SEM/TEM study, after various pre- and post-fixation regimes. Special attention was paid to the chorion, the contents of the peri-vitelline space, the plasma membrane, the syncytial layer, the blastodermal cells and the yolk. The preservation of these rather different structures with their varied chemical constituents was not straightforward. Standard fixation GA + OsO4 (glutaraldehyde, osmium tetroxide) did not adequately preserve these structures, and although well established non-coating techniques gave, with GA as prefixative, an acceptable image in FE-SEMI the preservation for TEM was inferior. The opposite result was obtained 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 much improved. Introduction of a mixture of GA, PF (paraformaldehyde), acrolein and picric acid as prefixative, in combination with the TAO (tannic acid/arginine/OsO4) non-coating post-fixation technique for FE-SEM and the OsO4/K4Fe(CN)(6) (osmium tetroxide/potassium ferrocyanide) post-fixation method for TEM? respectively, resulted in an adequate preservation of the chorion, plasma membrane, syncytial layer and cells of the blastoderm.