A method for expanding cuticle-enclosed intact C. elegans, while permitting readout of a majority of antigens that are detectable through non-IgM-class antibodies (~70%; estimated from the immunostaining results from the panel of 12 non-IgM antibodies in Figure 12A). Sample processing prior to Panel A is identical to the workflow for the standard ExCel protocol without ExFISH (as shown in blue arrows in Figure 1) until, and including, the gelation step (Figure 1A–C, E–G). The linear expansion factor of the hydrogel-specimen composite is shown in parentheses. For stages in which the worm tissue expands to a less extent than the surrounding hydrogel, which occurs due to incomplete homogenization of mechanical strength of the fixed worm tissue, the expansion factors of the worm and of the hydrogel are shown in front of and after a slash sign, respectively. (A–L) Steps of the protocol, with the bold text indicating the title of the step. (A) Hydrogel polymerization is performed on the specimen, by first incubating the specimens in activated monomer solution (0.015% 4-hydroxy-TEMPO, 0.2% TEMED, 0.2% APS, in addition to the non-activated monomer solution) for 1 hr at 4°C, transferring the specimens into a gelation chamber, and incubating the chamber for 2 hr at 37°C. During polymerization, AcX-modified proteins are covalently anchored to the hydrogel network. (B) Specimens are treated with chromatography-purified collagenase type VII at 0.5 kU/mL, in a calcium-containing tris-buffered saline (100 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2) overnight (18–24 hr) at 37°C. During this treatment, the hydrogel expands by ~1.2x linearly, whereas the worm slightly reduces in size to ~0.9x linearly. Due to the mismatch in expansion factor between the worm and the gel, the worm tissue detaches from the surrounding hydrogel, but physically remains in the hydrogel mold that was made of its own shape during the gelation step in A. (C) Specimens are processed with a denaturation treatment, in which they are incubated in a minimally-expanding protein-denaturing buffer (MAP5 buffer; 50 mM Tris pH 9.0, 5.72% (w/w) sodium dodecyl sulfate (SDS), 400 mM NaCl, 20 mM CaCl2) overnight (18–24 hr) at 37°C, overnight (18–24 hr) at 70°C, and 2 hr at 95°C. Reduced calcium and NaCl concentrations are used in this buffer, compared to other non-expanding buffers designed in this paper, due to their incompatible solubility with SDS at higher concentrations. (D) Specimens are washed four times in a tris-buffered saline (TNC40020 (acronyms are used in the supplementary protocols in Appendix 1) buffer; 50 mM Tris pH 8.0, 400 mM NaCl, 20 mM CaCl2) to remove SDS from the hydrogel sample. Specimens are then washed four times in tris buffered saline with reducing calcium concentration (once with TNT Buffer + 10 mM CaCl2, and then three times with TNT Buffer; TNT Buffer is 50 mM Tris pH 8.0, 1M NaCl, 0.1% Triton X-100) to remove calcium ions from the hydrogel sample. Finally, specimens are washed with phosphate-buffer saline with reducing NaCl concentration (once with PBST + 500 mM NaCl, twice with PBST; PBST is 1x PBS + 0.1% Triton X-100). (E) Specimens are immunostained with fluorescent antibodies against the target antigens. (F) Specimens are incubated with AcX at a concentration of 0.1 mg/mL in PBST (1x PBS + 0.1% Triton X-100) overnight at RT. This step equips proteins, including the fluorescent antibodies introduced in E, with a polymer-anchorable moiety. (G) Specimens are incubated in non-activated G2 monomer solution (50 mM MOPS pH 7.0, 2 M NaCl, 7.5% (w/w) sodium acrylate, 2.5% (w/w) acrylamide, 0.15% (w/w) N,N’-methylene-bis-acrylamide) overnight at 4°C, to ensure complete diffusion of the monomer solution throughout the specimen, prior to the gelation reaction. (H) Specimens are re-embedded into a second expandable hydrogel, by incubating the specimens in activated monomer solution (0.015% 4-hydroxy-TEMPO, 0.2% TEMED, 0.2% APS, in addition to the non-activated monomer solution) for 45 min at 4°C, transferring the specimens into a gelation chamber, and incubating the chamber for 2 hr at 37°C. During polymerization, AcX-modified fluorescent antibodies are covalently anchored to the hydrogel network of the second hydrogel (orange grids). We use blue grids to represent the hydrogel network of the first, DATD-crosslinked hydrogel (i.e., the network synthesized in Panel A), to differentiate it from the network of the re-embedding second hydrogel. (I) Specimens are treated with Proteinase K at 8 U/mL, in non-expanding digestion buffer (50 mM Tris pH 8.0, 500 mM NaCl, 40 mM CaCl2, 0.1% Triton X-100) overnight (18–24 hr) at RT, to further reduce the mechanical strength of the original worm tissue and permit greater expansion. During this proteolytic treatment, most proteins lose antigenicity, but some of the fluorescent signals from AcX-anchored fluorescent proteins are retained, as utilized by the original ProExM protocol. (J) Specimens are treated with DATD cleaving solution (20 mM sodium meta-periodate in 1x PBS, pH 5.5) for 30 min at RT, to chemically disintegrate the first hydrogel, which contains the periodate-cleavable crosslinker N,N'-diallyl-tartardiamide (DATD), while sparing the second hydrogel, which contains a periodate-resistant crosslinker, N,N’-methylene-bis-acrylamide (bis). (K) To visualize anatomical features, specimens can be stained with an N-hydroxysuccinimide ester (NHS ester) of fluorescent dye (introduced in Main Text, Figure 4, Videos 1 and 2). NHS-ester staining is performed at 5 μM in PBST (1x PBS + 0.1% Triton X-100) overnight at RT. DAPI staining can be applied at this stage, but the result does not correspond to the expected nuclear pattern as observed in Figures 2, 5 and 9 (see Figure 13—figure supplement 1). (L) Specimens are expanded with one wash in 0.1x PBS and two washes in deionized water. At this stage, the hydrogel expands by ~7.4x linearly, whereas the worm tissue expands by ~2.8x linearly, within a range from 2.5 to 3.5x (median, 2.78x; mean, 2.83x; n = 10 independently processed hydrogels from 2 sets of experiments). After expansion, specimens are ready for imaging.