Wild type Saccharomyces cerevisiae cells were high pressure frozen and embedded in HM20 resin. Samples were then stained for Nuclear Pore Complex (NPC) assosiated proteins with the monoclonal antibody ab24609 (Abcam) and visualized through transmission electron microscopy (TEM). The images show the nucleus of the cells and the black arrow indicates nuclear envelope budding events. The black gold particles represent the presence of NPC proteins.
Wild type cells were cultured in YPD medium and high pressure frozen in a Wohlwend Compact 3 machine, followed by a short freeze substitution protocol in 2% Uranyl acetate and embedded in HM20 resin. For the immuno-EM, samples were blocked for 2 hours in 1% BSA with 0.1% fish skin gelatine. A 1:50 dilution of the primary antibody ab24609 was used and a 1:150 dilution of the secondary antibody rabbit anti-mouse immunoglobulins (E0433, Agilend/Dako). Another incubation with a 1:70 diluted 10 nm gold-conjugated protein A (E1808, CMC UMC Utrecht) was followed. Finally, samples were post fixed in 2.5% glutaraldehyde for 20 minutes. Sections of 70 nm thickness were then contrast stained with 2% Uranyl acetate and Reynold's lead citrate. Pictures were aquired in a Tecnai T12 TEM with a Ceta CMOS 16M camera.