Excised tissue was primarily fixed with 4% v/v Formaldehyde / 0.5% Glutaraldehyde in sodium cacodylate buffer with 2uM of calcium chloride overnight, followed by several washes in same buffer. The samples were post-fixed with aqueous 1% w/v osmium tetroxide/ 1.5% w/v potassium ferrocyanide for 60 min, washed with distilled, de-ionised water, followed by a further 60 minutes in 1% w/v aqueous osmium. After further washes with DDW, the tissue was en bloc stained in 2% w/v aqueous uranyl acetate for 60 min. Following dehydration through an ethanol series, the samples were treated with several short washes through ice cold acetone, before gradual infiltration with Durcopan resin (Sigma-Aldrich UK). Once fully infiltrated with Durcupan, the tissue was embedded in BEEM capsules, and polymerised at 60oC for 48 h.
Thin sections, of approximately 80 nm thickness, were cut from each sample using a Leica UC7 ultramicrotome, collected on copper mesh grids and counter stained Reynolds’ lead citrate. Sections were observed using a JEOL JEM-1400 transmission electron microscope, using an accelerating voltage of 100 kV. Digital Images were recorded using a SIS Megaview III Digital Camera (Olympus Soft Imaging Solutions, Germany) with iTEM Software V 5.1 (Olympus, Germany).
Thin sections, of approximately 80 nm thickness, were cut from each sample using a Leica UC7 ultramicrotome, collected on copper mesh grids and counter stained Reynolds’ lead citrate. Sections were observed using a JEOL JEM-1400 transmission electron microscope, using an accelerating voltage of 100 kV. Digital Images were recorded using a SIS Megaview III Digital Camera (Olympus Soft Imaging Solutions, Germany) with iTEM Software V 5.1 (Olympus, Germany).