Protocol for applying expansion microscopy to the study of mammalian neuromuscular junctions

Figure 1. Preparation of gel and imaging chambers (A) A standard microscope slide is wrapped with Parafilm to serve as the base of the gel chamber, where polymerization occurs. (B) Small fragments of coverslip glass are used as spacers; one such piece is highlighted by a yellow box, while dashed boxes indicate the approximate positions where spacers are affixed. Yellow arrows denote the application of a droplet of water, which facilitates adhesion of the spacers to the slide. (C) A coverslip pre-coated with poly-L-lysine (yellow arrowhead) is affixed to the base using PinkySil silicone molding rubber. The silicone adhesive is prepared by mixing the pink and white components in a 1:1 ratio and applied along the edges of the coverslip to ensure secure attachment. (D) A custom acrylic chamber (yellow star) is subsequently positioned on top of the coverslip, and gentle pressure is applied to ensure complete and uniform adhesion to the underlying surface.

Figure 2. Muscle fiber preparation for ExM (A) Representative samples of dissected muscle tissue: mouse (top) and human (bottom). Both muscle types were freshly isolated and fixed in 4% PFA to preserve morphological integrity for whole-mount NMJ immunolabeling and subsequent ExM processing. (B) Microdissection of a human muscle sample. (C) The same teasing technique applied to mouse muscle samples. Individual fibers or small bundles were gently separated under a stereomicroscope using fine forceps to facilitate reagent penetration during staining and expansion. The yellow oval delineates the optimal size of teased fibers, ensuring sufficient tissue thinness for ExM protocol with preservation of native NMJ architecture. Scale bars represent 0.5 mm in (A, B and C). (D) Teased muscle fibers were transferred into individual wells of a 24-well plate, where the remaining steps of the ExM protocol were performed. This included immunostaining for NMJs, treatment with the anchoring agent (e.g., acryloyl-X, SE), and infiltration with monomer solution prior to gel polymerization and enzymatic digestion.

Figure 3. Embedding and gelation steps for ExM of muscle tissue (A) The teased muscle sample was carefully mounted onto a glass coverslip (18 × 18 mm, No. 1.5) previously coated with 250 μL of 0.1% poly-L-lysine solution for 1 hour to enhance tissue adhesion. The yellow dashed circle highlights the position of the muscle sample on the coverslip. (B) Gel polymerization (gelation) was initiated by sequential preparation of a 300 μL gelation mixture, as indicated by the black arrowheads. Of this mixture, 150 μL was applied into each gel chamber between two spacers (marked by red stars), yielding two individual expandable gels per chamber. (C) The tissue-mounted coverslip was gently inverted onto the gelation chamber, allowing contact between the muscle sample and the gel solution. Polymerization was carried out for 2 hours at 37°C in a humidified incubator. (D) Following gelation, the upper coverslip (to which the sample and gel adhered) was carefully separated from the chamber using a sterile blade. The spacers were simultaneously removed. (E) Schematic illustration of the gel detachment step, showing the muscle-bearing gel adhered to the upper coverslip. (F) The gel was trimmed with a…

Figure 4. Muscle sample digestion and gel expansion (A) After polymerization, gels (adhered to coverslips) were transferred to a six-well plate (one gel per well) and fully immersed in digestion buffer containing Proteinase K (1:100 dilution; 2–3 mL per well). Digestion was carried out at 37°C for 18–20 hours to ensure uniform expansion and minimize artefacts. (B) Schematic illustrating the digestion step. (C) Gels were then transferred to a 150 mm Petri dish and washed four times (30 min each) in ddH2O in darkness. After detaching from the coverslip and reaching stable size, gel dimensions were recorded to confirm 3.5–4× expansion. (D) Expanded gels were trimmed, oriented, and mounted using coverslips, metal spatulas, and a paintbrush. (E) Finally, transfer the gel into the imaging chamber (the custom acrylic chambers fitted with #1.5 coated coverslips).

Figure 5. Gel imaging and image processing (A) Imaging chambers containing the expanded gels were stored and transported in a microscope slide box to maintain darkness and minimize photobleaching. (B) Imaging was performed using an inverted confocal laser scanning microscope equipped with a 20×/0.75 NA air objective. Z-stacks were acquired at 0.5 μm intervals using a 16-bit, 512 × 512-pixel frame size at 9× digital zoom. Fluorescence excitation was set at 561 nm for the red channel and 488 nm for the green channel. (C) Confocal Z-stack images were deconvolved using Huygens software, and the resulting image stacks were imported into Fiji. Maximum intensity projections were generated for subsequent pre-processing and quantitative analysis.

Figure 6. Data analysis methods of expanded NMJs (A) Example of an expanded human NMJ stained with α-Bungarotoxin to label AChRs (cyan) and immunolabeled for SV2 to visualize presynaptic vesicles (magenta). Scale bar= 4 μm. A zoomed-in region (i) illustrates the selected area for measurement. AChR width was quantified on thresholded images (ii), indicated by red bars. AChR strip length and inter-strip spacing were measured on skeletonized images (iii), with yellow bars showing strip length and blue bars indicating spacing. Scale bar in (i)= 2 μm. (B) Mouse NMJ labeled as in (A). The yellow-dotted rectangle denotes the region used for fluorescence intensity profile analysis of AChRs and SV2. Intensity profiles were generated separately for each channel. Scale bar= 4 μm. Scale bar corresponds to the physical size of the expanded sample (not adjusted to expansion factor).