Improved immunostaining of nanostructures and cells in human brain specimens through expansion-mediated protein decrowding

Abstract

Proteins are densely packed in cells and tissues, where they form complex nanostructures. Expansion microscopy (ExM) variants have been used to separate proteins from each other in preserved biospecimens, improving antibody access to epitopes. Here, we present an ExM variant, decrowding expansion pathology (dExPath), that can expand proteins away from each other in human brain pathology specimens, including formalin-fixed paraffin-embedded (FFPE) clinical specimens. Immunostaining of dExPath-expanded specimens reveals, with nanoscale precision, previously unobserved cellular structures, as well as more continuous patterns of staining. This enhanced molecular staining results in observation of previously invisible disease marker-positive cell populations in human glioma specimens, with potential implications for tumor aggressiveness. dExPath results in improved fluorescence signals even as it eliminates lipofuscin-associated autofluorescence. Thus, this form of expansion-mediated protein decrowding may, through improved epitope access for antibodies, render immunohistochemistry more powerful in clinical science and, perhaps, diagnosis.

Fig. 1.. Decrowding expansion pathology (dExPath) for post-expansion immunostaining of human tissue and other formaldehyde-fixed specimens.

(A-E) Workflow for expanding formalin-fixed paraffin-embedded (FFPE) or formaldehyde-fixed human or mouse brain specimens. Key modifications of proExM/ExPath protocols are highlighted in green. PFA, paraformaldehyde; PBS, phosphate buffered saline; RT, room temperature; AcX, Acryloyl-X; SDS, sodium dodecyl sulfate. (A) Tissue samples undergo conversion into a state compatible with expansion. (B) Tissue samples are treated so that gel-anchorable groups are attached to proteins, then the sample is permeated with an expandable polyacrylate hydrogel. (C) Samples are incubated in a softening buffer to denature, and loosen disulfide bonds and fixation crosslinks between, proteins. (D) Softened samples are washed in a buffer to partially expand them. Linear expansion factor is shown in parentheses. (E) Samples are stained and then expanded fully by immersion in water.

Fig. 2.. Isotropy of dExPath.

(A-B) Representative pre-expansion super resolution structured illumination microscopy (SR-SIM) images of healthy human hippocampus (A) and human cerebrum high-grade glioma brain tumor tissue (B) which underwent processing as in fig. S3A with staining for MAP2 and DAPI (A), or for vimentin and DAPI (B). (C-D) Post-expansion images of the same fields of view as in (A-B), respectively. Samples underwent anchoring, gelation and softening (as in fig. S3B–C), another round of DAPI staining, ~4x linear expansion (as in fig. S3D), and imaging with confocal microscopy. (E-F) Root-mean-square (RMS) length measurement errors obtained by comparing pre- and post-expansion images such as in A-D (n = 4 samples, each from a different patient, E; n = 3 samples, each from a different patient, F). Line, mean; shaded area, standard deviation. Images are sum intensity z-projections, either of SR-SIM (A-B), or confocal (C-D) image stacks, both covering an equivalent tissue depth in biological units. Brightness and contrast settings: first set by the ImageJ auto-scaling function, and then manually adjusted to improve contrast for the stained structures of interest; quantitative analysis in (E-F) was conducted on raw image data. Scale bars (in biological units: physical sizes of expanded samples divided by their expansion factors, used throughout this manuscript, unless otherwise noted): (A-D) 5 μm. Linear expansion factors: (C-D) 4.0x.

Fig. 3.. dExPath removal of lipofuscin autofluorescence.

(A-C) Pre-expansion confocal images (single z slices) of a neuron in a a 5-μm-thick normal human cortex sample(format conversion as in Fig. 1A). Images were acquired for 3 fluorescent channel settings: (A) 488 nm ex/ 525 nm em; (B )561ex/607em; and (C) 640ex/685em. (D) Mean fluorescence intensities from pre-expansion images, averaged across regions of interest (ROIs) that exhibited prominent lipofuscin (left bar graph), as well as across background ROIs (right bar graph); (n = 4 tissue samples, each from a different patient). Brightness and contrast settings: first set by the ImageJ auto-scaling function, and then manually adjusted to improve contrast for lipofuscin; quantitative analysis was conducted on raw image data. Box plot: individual values (open circles; 3 measurements were acquired from each patient), median (middle line), mean (dotted line), first and third quartiles (lower and upper box boundaries), lower and upper raw values (whiskers). Statistical testing: 2-tailed paired t-test (non-Bonferroni corrected) was applied to lipofuscin vs. background, for pre-expansion mean fluorescence intensities for each spectral channel. *, p < 0.05; ns, not significant. (E-G) Shown are post-expansion confocal images after the sample from A-C was treated with anchoring, gelation,softening and decrowding (as in Fig. 1B–D), DAPI staining, and ~4x linear expansion, without post-decrowding immunostaining. Sum intensity z-projections of image stacks corresponding to the biological thickness of the original slice, taken under identical settings and of the same field of view as A-C and displayed under the same settings. (H) Mean fluorescence intensities, from post-expansion images, averaged across the same lipofuscin (left) and background (right) ROIs used in panel D. Plots and statistics as in D. (I-K) Confocal images as in (E-G), after post-decrowding immunostaining for MAP2 (microtubule-associated protein 2), giantin, and synaptophysin (labeled with antibodies in the same spectral ranges as indicated above A-C), as well as stained for DAPI (not shown; used for alignment), and then re-expanded to ~4x linear expansion. (L) Representative pre-expansion confocal image of a tissue sample of FFPE 5-μm-thick normal human hippocampus processed as in fig S3A. Pre-expansion immunostaining for MAP2 (488ex/525em) and GFAP (glial fibrillary acidic protein) (640ex/685em). Solid arrow indicates a region with lipofuscin aggregates (GFAP-like staining but found in a neuron); dashed arrow indicates MAP2 staining without lipofuscin; dotted arrow indicates GFAP staining. (M) Confocal image of the same field of view as (L). Tissues underwent softening and ~4x expansion, followed by decrowding, post-decrowding staining for MAP2 and GFAP, and expansion to ~4x (as in fig. S3B–F). Arrows, as in L. Scale bars (in biological units): (A, E, I) 7 μm; (L, M) 5 μm. Linear expansion factors: (E-G, I-K) 4.3x; (M) 4.1x.

Fig. 4.. dExPath-mediated protein decrowding reveals cells and structure not detected in pre-expansion staining forms of expansion microscopy.

(A) Representative pre-expansion confocal image (single z slice) of 5-μm-thick FFPE normal human hippocampal tissue.(Sample underwent processing as in fig. S3A and immunostaining for MAP2 and GFAP. White box in (i) marks a region with sparse and discontinuous signals that is shown magnified and in separate channels at the right (MAP2 in (ii) and GFAP in (iii)). MAP2 staining of a putative cell body (asterisk in (i)) and dendrite (upper dashed arrow in (i)). GFAP staining of a putative astrocytic process (lower dashed arrow in (i)) and discontinuous GFAP regions (dotted arrows in (iii)). Solid arrows show regions that were MAP2-negative (ii) or GFAP-negative (iii) in pre-expansion images (A), for comparison to post-expansion staining panels later in this figure. (B) Shown is a post-expansion confocal image after processing as in fig. S3B–D and imaging at ~4x linear expansion. Sum intensity z-projection of an image stack covering the biological thickness of the original slice (used for all expanded images throughout this figure); images were of the same fields of view as in (A), using identical hardware settings. Asterisks and arrows as in (A). (C) Post-decrowding stained confocal images of the same fields of view as in (A-B) after decrowding and additional immunostaining for MAP2 and GFAP and re-expansion to ~4x (fig. S3E–F), using identical hardware settings. Asterisks and arrows as in (A). (D) Quantification of fluorescence intensities for raw data of images post-expansion such as those of B (NR, “not restained”) and C (R, “restained”), averaged across MAP2-positive ROIs, for the MAP2 channel (cyan) and the GFAP channel (magenta). Box plot: individual values (open circles; 3 measurements were acquired from each patient), median (middle line), mean (dotted line), first and third quartiles (lower and upper box boundaries), lower and upper raw values (whiskers). Statistical testing: 2-tailed paired t-test (non-Bonferroni corrected) *, p < 0.05. (E) As in D, but for GFAP-positive ROIs, for the MAP2 channel (cyan) and the GFAP channel (magenta). (F) As in D, but for double negative ROIs, for the MAP2 channel (cyan) and the GFAP channel (magenta). (G) Representative pre-expansion confocal image (single z slice) of 5-μm-thick FFPE human high-grade glioma. Sample underwent format conversion, antigen retrieval, and immunostaining for GFAP and α-SMA (α-smooth muscle actin), and DAPI staining (fig. S3A). White box in (i) marks a region with sparse and discontinuous signals that is shown magnified and in separate channels at the right (GFAP in (ii) and α-SMA in (iii)). α-SMA-staining of pericytes that are enveloping blood vessels (dashed arrow in (i)). Discontinuous GFAP regions (dotted arrow in (ii)). Solid arrows in (i) and (ii) show regions that were GFAP-negative pre-expansion (G), for comparisons to post-expansion staining panels later in this figure. (H) As in B, but for panel G. (I) As in C, but for panel G. (J) As in D, but for the GFAP (cyan) and α-SMA (magenta) channels, in GFAP-positive ROIs. (K) As in D, but for the GFAP (cyan) and α-SMA (magenta) channels in α-SMA-positive ROIs. (L) As in D, but for the GFAP (cyan) and α-SMA (magenta) channels in double negative ROIs. (M) Representative (pre-expansion confocal image (single z slice) of 5-μm-thick human high grade glioma tissue (cortex or white matter). Sample underwent format conversion, antigen retrieval, and immunostaining for vimentin and α-SMA, and DAPI staining (fig. S3A). White box in (i) marks a region including part of a blood vessel that is shown magnified and in separate channels to the right (vimentin in (ii) and α-SMA in (iii)). Vimentin and α-SMA-staining of the blood vessel wall (dashed arrow in (i)) which surrounds the vessel lumen (asterisk in (i)). A vimentin-positive cell outside the blood vessel (dotted arrow in (i)). Solid arrows in (i) and (ii) show regions that were vimentin-negative pre-expansion (M), for comparison to post-expansion staining panels later in this figure. (N) As in B, but for panel M. (O) As in C, but for panel M. (P) As in D, but for the vimentin channel (cyan) and the α-SMA channel (magenta), in vimentin-positive ROIs. (Q) As in D, but for the vimentin channel (cyan) and the α-SMA channel (magenta), in α-SMA-positive ROIs. (R) As in D, but for the vimentin channel (cyan) and the α-SMA channel (magenta), in double negative ROIs. (S) Representative pre-expansion confocal image (single z slice) of 5-μm-thick human low grade glioma tissue (cortex or white matter). Sample underwent format conversion, antigen retrieval, and immunostaining for ionized calcium binding adapter molecule 1 (Iba1) and GFAP, and DAPI staining (fig. S3A). White box in (i) marks a region with sparse and discontinuous signals that is shown magnified and in separate channels to the right (Iba1 in (ii) and GFAP in (iii)). Iba1 staining of discontinuous regions (dotted arrow in (ii)). Solid arrows in (i) and (ii) show regions that were Iba1-negative pre-expansion (S), for comparison to post-expansion staining panels later in this figure. (T) As in B, but for panel S. (U) As in C, but for panel S. (V) As in D, but for the Iba1 channel (cyan) and the GFAP channel (magenta), in the Iba1-positive ROIs. (W) As in D, but for the Iba1 channel (cyan) and the GFAP channel (magenta), in GFAP-positive ROIs. (X) As in D, but for the Iba1 channel (cyan) and the GFAP channel (magenta), in the double negative ROIs. Scale bars: (A-C) panel i, 9 μm; ii, 1.7 μm; (G-I) i, 7 μm; ii, 0.7 μm; (M-O) i, 8 μm; ii, 0.8 μm; (S-U) i, 8 μm; ii, 0.8 μm. Linear expansion factors: (B,C) 4.1x; (H,I) 4.0x; (N,O) 4.3x; (T,U) 4.2x.

Fig. 5.. dExPath-mediated protein decrowding reveals cells and structures not detected by SR-SIM imaging of unexpanded tissues.

(A-B) Representative pre-expansion SR-SIM images of FFPE 5-μm-thick human tissue (processed as in Fig. S3A). (A) High-grade glioma tissue stained for vimentin and DAPI. Solid and dashed white boxes in (i) mark two separate regions shown magnified in (ii) (solid box) and (iii) (dashed box), respectively. Dotted arrows mark regions that appear as punctate and discontinuous in pre-expansion SR-SIM images for vimentin in (ii) and (iii), and solid arrows mark regions that were negative for vimentin in (iii), for comparison to post-expansion staining panels later in this figure. (B) Normal human hippocampus tissue stained for MAP2, GFAP and DAPI. (A) Solid white box in (i) shown magnified in (ii) for MAP2 and in (iii) for GFAP. Arrows as in (A) but for MAP2 and GFAP, in their respective images. (C-D) Shown are representative samples used for (A-B) after processing for post-expansion imaging Fig. S3B–D) and not restained. Sum intensity z-projection of an image stack covering the biological thickness of the original slice (used for all expanded images throughout this figure); images were of the same fields of view as in (A-B). Arrows as in (A-B). (E-F) Images of the same fields of view as in (A-B) after decrowding and additional restained for vimentin (E), or MAP2 and GFAP (F), followed by DAPI staining and re-expansion to ~4x (Fig. S3E–F), imaged using identical hardware settings as in (C-D). Arrows as in (A-B). Brightness and contrast settings in images (A-F): first set by the ImageJ auto-scaling function, and then manually adjusted to improve contrast for stained structures. Scale bars (in biological units): (A, C, E) left column, 8.3 μm; middle and right columns 840 nm; (B, D, F) left column, 6.0 μm; middle and right columns 500 nm. Linear expansion factors: (C) 4.1x; (D) 4.3x; (E) 4.1x; (F) 4.2x

Fig. 6.. dExPath removes autofluorescence from amyloid plaques and preserves detection of disease markers in Alzheimer’s disease (AD).

(A) Representative pre-expansion confocal image (single z slice) of an amyloid β plaque within a FFPE 5-μm-thick sample of AD human cortex. The samples underwent processing as in Fig. 1A. (i) Images were acquired for the fluorescent channel setting of 488ex/525em. Solid arrow points to an amyloid β plaque. (ii) Mean fluorescence intensities from pre-expansion images, averaged across regions of interest (ROIs) taken at amyloid β plaque ([plaque], left bar) and background ROIs ([Bck], right bar); Brightness and contrast settings: first set by the ImageJ auto-scaling function, then manually adjusted to improve contrast for amyloid β plaque; quantitative analysis in ii was conducted on raw image data. Box plot: individual values (open circles; 3 plaque measurements were acquired from each patient), median (middle line), mean (dotted line), first and third quartiles (lower and upper box boundaries), lower and upper raw values (whiskers). Statistical testing: 2-tailed paired t-test was applied to amyloid β plaque vs. background, for pre-expansion mean fluorescence intensities. *, p < 0.05; ns, not significant. (B) Post-expansion confocal images after the sample from A was processed as in Fig. 1B–D, post-decrowding methoxy-x04 stained, and ~4x linear expansion. Images were acquired for 2 common fluorescent channel settings: (i) a 405ex/450em channel to detect methoxy-x04; and (ii) a 488ex/525em channel to detect plaque autofluorescence as in A. Sum intensity z-projections of image stacks corresponding to the biological thickness of the original slice, taken under identical settings and of the same field of view as in A and displayed under the same settings. (iii) Mean fluorescence intensities, from post-expansion images, averaged across the same amyloid β plaque (left bar) and background (right bar) ROIs used in A. Plots and statistics as in A. (C) Images of the same field of view as in (A-B), but the sample was additionally immunostained post-decrowding (as in Fig. 1E), with a (i) Aβ(–42) (amyloid β protein) monoclonal antibody and images were acquired for the channel settings 561ex/607em channel and (ii) methoxy-x04 using the same spectral ranges as indicated in B at ~2.2x linear expansion; brightness and contrast settings adjusted as in (A) to improve contrast for stained structures. (D) Confocal image of a FFPE 5-μm-thick sample of AD human cortex. Sample was processed as in Fig. 1A–D, post-decrowding immunostained, and imaged at ~2.3x linear expansion (Fig. 1E). The tissue sample was stained for Aβ(–42) (an amyloid β plaque marker) and phospho-tau (a neurofibrillary tangle marker), and GFAP (an astrocyte marker). White boxes mark regions shown magnified in insets on the right. All images are sum intensity z-projections of a confocal image stack. Brightness and contrast settings determined as in C. Scale bars (in biological units): (A - C) 25 μm. Linear expansion factors: (B) 4.1x; (C) 2.2x. Scale bars (in physical units): (D) left panel, 25 μm; inset, 10 μm. Linear expansion factor: (D) 2.3x.

Fig. 7.. dExPath reveals previously undetected cells defined by single and multiple markers of importance to glioma biology.

(A) Representative pre-expansion confocal image (single z slice) of a 5-μm-thick FFPE human low-grade glioma specimen. Sample was immunostained for vimentin, Iba1 and GFAP, and DAPI staining (Fig. S3A). Left panel, overlay of all 4 channels; right three panels, individual channels (not including DAPI). Dotted arrows show regions that were vimentin and GFAP negative in pre-expansion images, and solid arrows show regions that were Iba1, GFAP and vimentin negative in pre-expansion images, for comparison to post-decrowding staining panels later in this figure. (B) Sample used for (A) after anchoring, gelation), softening (fig. S3B–C), washing with PBS (which results in an expansion factor of ~2.3x), tissue shrinkage (via adding salt) to ~1.3x of the original size, and imaging. Single z slice image centered at the same midpoint of the original slice; images were of the same field of view as in (A), using identical hardware and software settings. Arrows as in (A). (C) Sample used for (B) after expansion (fig. S3D) for imaging at ~4x linear expansion. Sum intensity z-projection of an image stack covering the biological thickness of the original slice; images were of the same field of view as in (A), using identical hardware and software settings. Arrows as in (A). (D) Post-decrowding stained confocal images of the same field of view as in (A) after decrowding and additional immunostaining for vimentin, Iba1, and GFAP, tissue shrinkage (fig. S3E–F) and imaging at shrunken state. Arrows as in (A). (E) Sample used for (D) after expansion (fig. S3D) for imaging at ~4x linear expansion. Arrows as in (A). (F) Pixel level analysis of the percent of single or double positive stained pixels, from pre-expansion (gray boxes) and post-decrowding at shrunken state (white boxes) images, for vimentin (V), Iba1 (I), GFAP (G), Iba1 and vimentin (I&V), vimentin and GFAP (V&G), and Iba1 and GFAP (I&G). Values represent the percentage of positive pixels among all pixels in the field of view with 3 different values per sample each corresponding to a different field of view Box plot: individual values (open circles; 3 measurements were acquired from each patient), median (middle line), mean (dotted line), first and third quartiles (lower and upper box boundaries), lower and upper raw values (whiskers), used throughout the graphs of this figure. (G) Cell level analysis of single or double positive labeled cells, from pre-expansion and post-decrowding at shrunken state images. Values represent total number of labeled cells in the field of view. (H) Cell level analysis of the percentage of double positive labeled cells divided by all single positive cells for a stain in the pre-expansion and post-decrowding at shrunken state images. Values represent the percentage (%) of double positive cells relative to the total number of single positive cells for a stain. Brightness and contrast settings in images (A-E): first set by the ImageJ auto-scaling function, and then manually adjusted (by raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for stained structures but quantitative analysis in (F-H) was conducted on raw image data. Statistical testing: 2-tailed paired t-test (non-Bonferroni corrected) were applied on pre-expansion and post-decrowding values. *, p < 0.05; ns, not significant. Scale bars: (A-E) 11 μm. Linear expansion factors: (B, D) 1.3x; (C, E) 4.4x.