Fig. 4: pan-ExM-t is compatible with antibody labeling of brain tissue structural markers.

a, Cortical neurons pan-stained with NHS ester (grayscale) and stained with SYTOX Green (teal) showing a neuron nucleus. b–d, Magnified view of the area in the green box in a (b) and the SYTOX Green (c) and NHS ester (d) channels shown separately. e, Astrocyte pan-stained with NHS ester (grayscale) and immunolabeled with GFAP antibody (α-GFAP; magenta). f–h, Magnified view of the area in the magenta box in e (f) showing GFAP filaments surrounding mitochondria, and the α-GFAP (g) and NHS ester (h) channels shown separately. i, 3D rendering of α-GFAP (magenta), SYTOX Green (green) and NHS ester (grayscale). j,k, Two example 3D renderings of α-GFAP (magenta) and blood vessels (BV) pan-stained with NHS ester (grayscale). l, Multiciliated ependymal epithelia pan-stained with NHS ester (grayscale). Inset: ninefold symmetry of cilia. m–r, Lateral (m–o) and top (p–r) views of axons pan-stained with NHS ester (grayscale) and immunolabeled with MBP antibody (α-MBP; red). α-MBP (n,q) and NHS ester (o,r) channels of the areas shown in m and p. s, 3D rendering of anti-MBP showing multiple axons. t,u, Neuropil pan-stained with NHS ester (grayscale) and anti-GFP (green) showing that only a sparse subset of neurites (t) and cell bodies (u) express GFP-Thy1. v–x, Magnified area in the black box in t (v) showing a neurite pan-stained with NHS ester (x) and immunolabeled with anti-GFP (w) where GFP-Thy1 is not expressed inside mitochondria. y–gg, A dendritic spine (y–aa) and axonal boutons (bb–gg) pan-stained with NHS ester and immunolabeled with anti-GFP. z,cc,ff, Anti-GFP channels of the areas shown in y, bb and ee, respectively. aa,dd,gg, NHS ester channels of the areas shown in y, bb and ee, respectively. hh, 3D representation of a surface-rendered dendrite immunolabeled with anti-GFP (green) and NHS ester pan staining (grayscale). ii, 3D rendering of a dendrite immunolabeled with anti-GFP (green) and NHS ester pan staining (grayscale). Panels showing pan staining are displayed with a white-to-black color table; panels showing immunolabeling or SYTOX Green staining are displayed with a black-to-white color table. Gamma corrections: a,e,m,p,t,u,x,aa,dd,gg, γ = 0.7. 3D renderings were processed with Imaris with Gamma corrections applied. 3D image processing details for images i–l, s, hh and ii are found in the Methods section. Scale bars in the 3D renderings i–l, s, hh and ii are not corrected for the expansion factor. All other scale bars are corrected for the expansion factor. Scale bars: 5 µm (a), 2 µm (b–d,e,m–o,u), 500 nm (f–h,v–x), 1 µm (p–r,t), 200 nm (y–gg). Expansion factor: 22 ± 3.