Cells were washed in PBS and then fixed in PFA 4% in PBS for 20 min at room temperature. After rinsing with PBS, cells were put into a sucrose gradient to reach 30% sucrose overnight. Cells were then frozen in liquid nitrogen and immediately thawed at room temperature. Immunostaining was carried out without permeabilization step, directly with primary antibodies (rabbit anti-PATJ 1/100, for 3 hr at room temperature). After the washing steps, cells were incubated with a secondary antibody carrying 6 nm gold particles (goat anti-rabbit 1/20, 806.011, Aurion, The Netherlands). A tertiary antibody was used to observe where gold particles were localized on a macroscopic level (Alexa 568 conjugated donkey anti-goat 1/200 from Invitrogen, for 1 hr at room temperature).
Cells were then prepared specifically for electron microscopy. They were fixed in 2.5% glutaraldehyde, 2% PFA, and 0.1% tannic acid in sodium cacodylate 0.1 M solution for 30 min at room temperature. After washing steps, cells were post-fixed in 1% osmium in sodium cacodylate 0.1 M solution for 30 min at room temperature and contrasted in 2% uranyl acetate in water solution for 30 min at room temperature. Cells were then dehydrated in ethanol and embedded in Epon epoxy resin.
Cells were then prepared specifically for electron microscopy. They were fixed in 2.5% glutaraldehyde, 2% PFA, and 0.1% tannic acid in sodium cacodylate 0.1 M solution for 30 min at room temperature. After washing steps, cells were post-fixed in 1% osmium in sodium cacodylate 0.1 M solution for 30 min at room temperature and contrasted in 2% uranyl acetate in water solution for 30 min at room temperature. Cells were then dehydrated in ethanol and embedded in Epon epoxy resin.