Journal: The Journal of Cell Biology

Article Title: Label-retention expansion microscopy

doi: 10.1083/jcb.202105067

Figure Lengend Snippet: Workflow and characterization of LR-ExM. (A) Workflow of LR-ExM. (B) Schematic of trifunctional anchors. (C–E) ExM confocal images of CCPs in U2OS cells indirectly immunostained for clathrin heavy-chain (POI). (C) ProExM using AF488-conjugated secondary antibodies. (D) ExM with postexpansion labeling using biotin-conjugated antibodies. (E) LR-ExM using antibodies conjugated with NHS-MA-biotin trifunctional anchor. Samples in D and E were postexpansion stained with streptavidin-AF488. (F) Intensity quantification of C–E. Error bars represent SD. n = 3 for each case. (G) LR-ExM confocal image of CCPs in U2OS cells immunostained indirectly with secondary antibodies conjugated with NHS-MA-DIG anchor, postexpansion stained with anti-DIG antibody. (H and I) Cross sections of the CCP in the boxed area of G. The length expansion ratios for images in C, D, E, and G–I are 4.3, 4.5, 4.6, and 4.3, respectively. The length expansion ratio for the samples used in plot F is 4.5 ± 0.2. Scale bars, 500 nm (C–E and G) and 100 nm (H and I). All scale bars are in preexpansion units. arb. u., arbitrary units; STV, streptavidin.

Article Snippet: U2OS cells (ATCC; HTB-96) were cultured in McCoy's 5a (ATCC; 30–2007) supplemented with 10% FBS at 37°C in 5% CO 2 .

Techniques: Labeling, Staining

Journal: The Journal of Cell Biology

Article Title: Label-retention expansion microscopy

doi: 10.1083/jcb.202105067

Figure Lengend Snippet: Two-color LR-ExM images using immunostaining and protein tag approaches . (A) Two-color LR-ExM confocal image of microtubules labeled with NHS-MA-biotin–conjugated secondary antibodies (magenta) and CCPs labeled with NHS-MA-DIG–conjugated secondary antibodies (green) in a U2OS cell. (B) Magnified view. (C–E) LR-ExM confocal images of CCPs and/or mitochondria in HeLa cells labeled using SNAP tag–labeled clathrin (C), CLIP tag–labeled TOMM20 (D), and two-color imaging (E). (F) Magnified view. (G) LR-ExM confocal image of mouse brain slice indirectly immunostained for the presynaptic marker Bassoon (magenta) and the postsynaptic marker Homer1 (green). (H and I) Zoomed-in images of synapses. (J and K) Transverse intensity profiles along the yellow box long axes. Bassoon is labeled with NHS-MA-DIG–conjugated secondary antibodies, and Homer1 is labeled with NHS-MA-biotin–conjugated secondary antibodies. All samples are postexpansion stained with streptavindin-AF488 and or anti-Digoxin-AF594. The length expansion ratios for images in A and B; C; D; E and F; and G–I are 4.7, 4.4, 4.4, 4.5, and 4.2, respectively. Scale bars, 1 µm (A and G), 200 nm (B, F, H, and I), and 500 nm (C–E). All scale bars are in preexpansion units.

Article Snippet: U2OS cells (ATCC; HTB-96) were cultured in McCoy's 5a (ATCC; 30–2007) supplemented with 10% FBS at 37°C in 5% CO 2 .

Techniques: Immunostaining, Labeling, Imaging, Slice Preparation, Marker, Staining

Journal: The Journal of Cell Biology

Article Title: Label-retention expansion microscopy

doi: 10.1083/jcb.202105067

Figure Lengend Snippet: LR-ExM reveals subcellular protein organizations. (A) Two-color confocal LR-ExM of SNAP-tagged lamin A/C (cyan) and immunostained NPC (red hot) of a HeLa cell. (B) Magnified view. (C and D) Views of individual channels of B. Note the cytoplasmic background in A is caused by the anti-NUP153 antibody. (E) Histogram of lamin hole area in the boxed region. (F–I) Two-color confocal LR-ExM of SNAP-tagged lamin A/C (cyan) and immunostained H3K9me3 (magenta) of a HeLa cell, with a maximum intensity project of a z stack covering the bottom half of the nucleus (F), a single section of the nucleus (G), and magnified views of the boxed regions in G (H and I). (J–M) Two-color confocal LR-ExM of SNAP-tagged lamin A/C (cyan) and immunostained H3K4me3 (red), with a maximum intensity projection of a z stack covering the bottom half of the nucleus (J), a single section of the nucleus (K), and magnified views (L and M) of the boxed regions in K. (N) Correlation coefficients of NPC with lamin A/C, H3K9me3 with lamin A/C, and H3K4me3 with lamin A/C. (O–R) Confocal LR-ExM of lamin A/C in U2OS (O), HeLa (P), HEK 293T (Q), and mouse embryonic stem cells (mESC; R) showing maximum intensity projections over the bottom half of a nucleus. The length expansion ratios for images in A–D, F–I, J–M, O, P, Q, and R are 4.5, 4.5, 4.3, 4.2, 4.3, 4.6, and 4.4, respectively. Scale bars, 2 µm (A, F, G, J, K, and O–R) and 500 nm (B–D, H, I, L, and M). All scale bars are in preexpansion units.

Article Snippet: U2OS cells (ATCC; HTB-96) were cultured in McCoy's 5a (ATCC; 30–2007) supplemented with 10% FBS at 37°C in 5% CO 2 .

Techniques:

Journal: The Journal of Cell Biology

Article Title: Label-retention expansion microscopy

doi: 10.1083/jcb.202105067

Figure Lengend Snippet: LR-ExSIM of microtubules. (A) LR-ExSIM image of microtubules in a U2OS cell stained with antibody conjugated with NHS-MA-DIG anchors. (B) Magnification of A. (C) The transverse profile of the microtubule in the gold box in B. (D) Schematic of the structure of an immunostained microtubule. By fitting the peaks to Gaussian functions, we calculated the resolution (FWHM) of LR-ExSIM to be 34 nm. (E) LR-ExSIM image of Cep164 in distal appendages of a primary cilium of an expanded mouse embryonic fibroblast indirectly immunostained with NHS-MA-biotin secondary antibodies. The length expansion ratio is 4.2. (F) Magnified view of E. (G) STORM image of Cep164 in distal appendages of motile cilia of an unexpanded multiciliated mouse tracheal epithelial cell. (H) Magnified view of G. LR-ExSIM (E and F) and STORM (G and H) reveal structure of distal appendages with similar super resolution. The same primary antibody was used for both images. Scale bars, 1 µm (A), 500 nm (B), 2 µm (E and G), and 100 nm (F and H).

Article Snippet: U2OS cells (ATCC; HTB-96) were cultured in McCoy's 5a (ATCC; 30–2007) supplemented with 10% FBS at 37°C in 5% CO 2 .

Techniques: Staining