Mouse anti beta tubulin antibody

Cryo thin sections collected onto TEM grids were incubated 30 min on gelatin 2% in 0.1 M Sorensen buffer, pH 7.4, at 37 °C to remove the 12% gelatin (used for the embedding of the sample) from the section, then incubated 5 min in PBS buffer containing 1% BSA (Aurion, The Netherlands) as a blocking step to avoid nonspecific labeling. Sections were floated for 1 h on a drop of mouse anti-beta-tubulin antibody (Sigma-Aldrich, Switzerland) diluted at 1/50 in PBS containing 1% BSA. After washing with 0.1% BSA in PBS, the samples were incubated for 1 h with 10 nm colloidal gold-conjugated secondary antibodies, goat anti-mouse (Aurion, The Netherlands) diluted 1:30 in 1% BSA/PBS. The ultrathin cryo sections were then fixed in 1% glutaraldehyde in PBS for 10 min to further stabilize them, followed by eight washes in distilled water, 2 min each. Finally, samples were dried and PVA embedding was done as will be described in the subsection “Drying and embedding of the cryo sections for NanoSIMS analysis”.