Formvar coated

Sample primary fixation was done by 4% PFA, 2% Glutaraldehyde (Carl Roth #4157) in 0.1M Cacodylate Buffer (Science Services #11650). For transmission electron microscopy (TEM), the tissue was then post-fixed in 0.5% Osmium tetroxide (Science Services #E19150) in ddH₂O for 60min on ice and then washed 6x in ddH₂O. The tissue was incubated in 1% aqueous Uranyl acetate solution (Science Services #E22400–1) for 2h in the dark and washed 2x in ddH₂O. Dehydration was performed by 15min incubation steps in 30%, 50%, 70%, 90% and 2× 100% Ethanol (Fisher Scientific #32205) and 2× 100% Aceton (Sigma-Aldrich #179124). After embedding in Durcupan resin (Sigma-Aldrich #44611 and #44612), ultrathin sections (55nm) were performed using a UC7 Ultramicrotome (Leica), collected on Formvar-coated (Science Services #E15830–25) copper grids (Plano #G2500C). Post-staining was done for 1min with 3% Lead Citrate (Delta Microscopies #11300). For scanning electron microscopy (SEM), the fixated samples were dehydrated in 70%, 80%, 90% and 100% Ethanol (each step for 1h at RT) and incubated in a 1:1 solution of Ethanol and Hexamethyldisilazan (HMDS; Carl Roth #3840.2) for 30min. After incubation in 100% HMDS, the solvent was allowed to evaporate. The dehydrated tissue was mounted onto sample holders and sputtered with gold using a Polaron Cool Sputter Coater E 5100.