Co-Immunogold-labeling of A- and B-type lamins (a) Nuclei on EM-grids were treated with α-lamin A/C and α-lamin B1 and labeled with 6 nm and 10 nm gold conjugates, respectively, prior to vitrification and cryo-ET analysis. Control samples (middle and right) were either treated with α-lamin A/C prior to treatment with 6 nm gold conjugate, post-fixation and subsequent treatment with 10 nm gold conjugate (middle panel), or only with 6 nm and 10 nm gold conjugate (right panel), omitting incubation with antibodies. Large projection views of parts of nuclei (contoured by the light-grey line) display the distribution of gold conjugates under the indicated conditions. Scale bar, 200 nm. (b) Box plots show the labeling density of 6 nm (lamin A/C) and 10 nm (lamin B1) gold particles per μm 2  (+/− SD) under the indicated conditions (white line represents the median and black dot the average amount of gold particles). The number of gold particles was extracted from 47 sub-volumes (left panel), 9 sub-volumes (middle panel) or 11 sub-volumes (right panel). (c) To validate the reliable assignment of small and large gold colloids, 6 nm and 10 nm gold conjugated protein A label were manually picked from samples containing either 6 nm or 10 nm colloids (individual gold colloids), a mixture of both (mixed gold colloids) or from the co-immunogold labeling experiment (lamin A/C and B1 co-labeling experiments) and averaged (n = 200 per condition). The two images at the bottom show a collage of nine individual 6 nm (left) and 10 nm (right) gold conjugated protein A label, respectively, which were picked from the co-immunogold labeling experiments. The line plot shows the normalized intensity profile of the average diameter of the gold conjugated protein A labels that were picked from the indicated samples. The solid lines show the intensity profile of the averaged gold label from the samples containing only one type of colloid. The dashed lines show the intensity profile of the averaged gold labels picked from a mixture. The lines with diamonds show the profile of the averaged gold labels from the co-immunogold labeling experiment. The line profiles of the 6 nm and 10 nm averaged gold conjugated protein A labels are shown in green and red, respectively. The line plot shows almost identical diameters of the 6 nm or 10 nm gold labels for each condition.