Article Title: Expansion microscopy of C. elegans
Figure Lengend Snippet: Screen of post-gelation treatments that confer tissue expandability and general stainability of epitopes. ( A ) Representative transillumination images of paraformaldehyde-fixed, β-mercaptoethanol-reduced, AcX-treated, and hydrogel-embedded hermaphrodite animals, (left column) right after hydrogel embedding and prior to any hydrogel expansion, (middle column) after 1.9x-2.1x hydrogel expansion, by incubating the gelled sample in 1x PBS, or (right column) after 3.3–3.7x hydrogel expansion, by sequentially washing the gelled sample with 0.5x PBS, 0.1x PBS, and 0.01x PBS. After hydrogel embedding, gelled samples either are left in TNT buffer (top row; no treatment), processed with a 2 day 37°C Proteinase K digestion, as in the standard ExCel protocol (middle row), or processed with a 5 day 37°C collagenase type II digestion (bottom row). Transillumination images provide visualization to both the contour of the worm (traced under high digital magnification in black dotted lines, in cases where direct observation is difficult due to reduced tissue scattering after hydrogel expansion), and also the contour of the mold in the embedding hydrogel (traced in white dotted lines, in cases where direct observation is difficult to reduced gel-boundary scattering after hydrogel expansion). For each treatment, the expansion factor of the worm (measured as the length ratio of a worm in the pre-expansion and the post-3.5x-expansion (hydrogel) state) is normalized by the expansion factor of the embedding hydrogel, which results in a normalized expansion factor (abbreviated as nExF), to remove the variation on worm expansion factor due to inter-sample variation in the hydrogel expansion factor. For the no-treatment condition (top row) and the collagenase type II condition (bottom row), where the normalized expansion factors are markedly less than unity (0.40 and 0.66, respectively), the hydrogel-embedded worm tissue detaches from the surrounding hydrogel, due to tissue mechanical hindrances against expansion that are incompletely removed by the post-hydrogel-embedding treatment, and can be visualized by the extent of mismatch between the worm contour and the hydrogel-mold contour. Images are single-plane wide-field acquisitions. For post-2.0x- and 3.5x- images, in cases where uneven illumination from the bright-field light source strongly affects contour visualization, a band-pass filtering with the boundary of 3 and 30 pixels was performed with the Fiji function ‘Bandpass Filter’ to remove the illumination artifact, and to improve contour visualization. Brightness and contrast settings: first set by the automatic adjustment function in Fiji, and then manually adjusted (raising the minimum-intensity threshold and lowering the maximum-intensity threshold) to improve contrast for the boundaries of the worm and the mold. Scale bars: 300 μm in actual units (not converting to biological units here, since the two features (worm and hydrogel-mold) are associated with different expansion factors). ( B ) Representative images of the immunostaining of hydrogel-embedded animals in ( A ), via a panel of 5 primary antibodies with known patterns of staining. Due to spectral limitation, the five antibodies were separated into four spectrally separable channels (DyLight 405 for anti-GFP, Alexa 488 for anti-LMN1, Alexa 546 for anti-myotactin and anti-DLG1, Alexa 647 for anti-acetylated tubulin). An IHC score from 0 to 1 was manually assigned to each channel, based on the estimated signal-to-noise ratio of the expected pattern of staining, and thereby provides a rough quantification for the quality of immunostaining of each antibody (or pair of antibodies) following the specified post-hydrogel-embedding treatment. The strain used had pan-neuronal cytosolic expression of GFP ( tag-168p::GFP ). A few patterns of channel crosstalk, such as the anti-GFP signal observed in the anti-myotactin + anti-DLG1 channel, were observed but do not affect the scoring process, because the known patterns of staining for each of the five antibodies were spatially separable (GFP, pan-neuronal by promoter choice; LMN1 (lamin), nuclear; myotactin, periphery of pharyngeal muscle and beneath cuticle; DLG1 (disc large), adherens junctions that form characteristic thread-like patterns across the length of the worm; acetylated tubulin, touch-receptor neurons). Images are max-intensity projections of confocal stacks acquired through the entire animal. Brightness and contrast settings: individually set by the automatic adjustment function in Fiji. Linear expansion factors of the hydrogel: 1.9–2.1x (after immunostaining, the samples were left in 1x PBS and imaged in that state, without further expansion in deionized water; we decided to use this procedure here, because we observed that even at this partially expanded state, we could already evaluate whether the staining against protein targets yielded the expected patterns of localization, as demonstrated by the images in this panel, without the additional improvements in resolution that would result from further expansion of the samples). Linear expansion factors of the worm: no treatment, 1.1x; Proteinase K (standard ExCel), 1.9x, Collagenase Type II, 1.6x. Scale bars: left images, 50 μm (in biological units, i.e. post-expansion lengths are divided by the expansion factor of the worm). ( C ) Summary of the screen of 22 post-hydrogel-embedding treatments, each of which is characterized by (X axis) the post-treatment expandability of the worms, as quantified by the normalized expansion factor analysis as performed in A, and (Y axis) the post-treatment quality of immunohistochemistry, as quantified by the average of IHC scores across the four channels in the immunostaining assay as performed in B. Each dot represents a single treatment. See Methods for the protocol performed for each treatment. Treatments are grouped based on the nature of the protocol, and colored according to the group they belong to (legend). X- and Y- coordinates of each treatment represent the mean values of all animals analyzed in the expandability assay (which quantifies the normalized expansion factor, as in A) and the immunostaining assay (which quantifies the IHC score, as in B), respectively. Number of animals analyzed in the assay: expandability assay, 3–4 animals from 1 hydrogel sample; 4-channel immunostaining assay, 2–4 animals from 1 hydrogel sample, except for the papain treatment (1 animal). The condition displayed as MAP5 18-18-2 (heat denaturation in MAP5 buffer for 18 hr at 37°C, 18 hr at 70°C, and 2 hr at 95°C) is abbreviated as simply ‘MAP5’ in later figures. Source data of the measurements made in the expandability and the stainability assays are available in Figure 11—source data 1 . Measurements for the expandabiliy and stainability assays, whose population statistics are summarized in Figure 11C .
Article Snippet: Standard ExCel: Proteinase K digestion Proteinase K (New England Biolabs, 800 U/mL stock) was diluted at 1:100 into non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2 , 0.1% Triton X-100).
Techniques: Immunostaining, Staining, Immunohistochemistry, Expressing