Diversity in Nuclear Pore Complexes Figure 10. High resolution STED microscopy resolves NPC clusters, reveals microheterogeneity in NPC populations, and demonstrates that NPCs from different populations are similar in size. HIgh resolution STED microscopy (STED) and confocal (Conf.) double immunofluorescence microscopy were performed with NUP62 (green) and NUP214 (red) antibodies in S12 neuroblastoma cells. All images are projections of 8–10 um total optical z-stacks. Bars represent 500 nm. A. Comparison of confocal and STED microscopy resolution of NUP214 antibody immunofluorescence. A and B. Arrows, NUP62+/NUP2142 NPCs; asterisks, NUP62.NUP214 NPCs; diamonds, NUP62+/NUP214+ NPCs; triangles, NUP62,NUP214s; arrowheads, NUP622/NUP214+ NPCs. C, D, and E. Measurement of diameters of NUP62+/NUP2142 (C), NUP622/NUP214+ (D), and NUP62+/NUP214+ (E) NPCs in S12 cells. Measured pores were derived from STED microscopy images (indicated by asterisks). Yellow represents overlap between green and red. doi:10.1371/journal.pone.0036137.g010 from Thermo Scientific (product number MA1-81617). All secondary antibodies were purchased from Jackson ImmunoR- esearch Laboratories. Immunofluorescence microscopy Subconfluent cells were cultured on glass coverslips. Immuno- fluorescence staining was performed similarly to Maeshima et.al [64]. Cells were fixed with 3.7% paraformaldehyde in PBS and quenched with 50 mM glycine in HMK buffer (20 mM HEPES; pH 7.5, 1 mM MgCl2, 100 mM KCl). Coverslips were washed with HMK briefly. After cells were treated with 0.5% Triton X- 100 in HMK, they were treated with blocking solution (10% normal donkey serum and 1% BSA in HMK buffer). Cells were incubated with primary antibody for 1 to 2 hours at room temperature. After washing with HMK five times, the cells were incubated with secondary antibodies (Jackson ImmunoResearch). PLoS ONE | www.plosone.org 12 April 2012 | Volume 7 | Issue 4 | e36137