For SEM imaging embryos were fixed in 4% PFA in 1X PBS and processed for SEM as previously described in Jongebloed et al. (1999) (link) using tannic acid, osmium tetroxide, thiocarbohydrazide and osmium tetroxide (TOTO) to enhance conductivity before dehydration through a graded series of ethanol, followed by drying in a Tousimis Samdri 795 critical point dryer. Dried samples were then mounted on stubs, coated with 4 nm gold palladium in a Leica ACE600 coater and imaged in a Zeiss Merlin SEM at 20 kV with SE2 detector.
For TEM analysis, embryos were prefixed with 2.5% paraformaldehyde and 2% glutaraldehyde in 50 mM sodium cacodylate containing 1% sucrose (pH 7.4). The samples were post fixed in 2% OsO4. After dehydration through a graded ethanol series, samples were infiltrated and embedded in Epon resin (EMS, Fort Washington, PA). Ultrathin (60–80 nm) sections were cut with a diamond knife and collected on single-slot copper grids. Grids were post-stained with 2% uranyl acetate and 1% lead citrate. Images were acquired on a FEI transmission electron microscope (Tecnai Bio-TWIN12, FEI) at 80 kV.
For TEM analysis, embryos were prefixed with 2.5% paraformaldehyde and 2% glutaraldehyde in 50 mM sodium cacodylate containing 1% sucrose (pH 7.4). The samples were post fixed in 2% OsO4. After dehydration through a graded ethanol series, samples were infiltrated and embedded in Epon resin (EMS, Fort Washington, PA). Ultrathin (60–80 nm) sections were cut with a diamond knife and collected on single-slot copper grids. Grids were post-stained with 2% uranyl acetate and 1% lead citrate. Images were acquired on a FEI transmission electron microscope (Tecnai Bio-TWIN12, FEI) at 80 kV.