For AcX anchored fluorescent proteins and antibody staining, the following steps – gelation, digestion and expansion – can be performed as described previously1 . Briefly, monomer solution (1× PBS, 2 M NaCl, 8.625% (w/w) sodium acrylate, 2.5% (w/w) acrylamide, 0.15% (w/w) N,N′-methylenebisacrylamide) was mixed, frozen in aliquots, and thawed before use. Monomer solution was cooled to 4°C before use. Concentrated stocks (10% w/w) of ammonium persulfate (APS) initiator and tetramethylethylenediamine (TEMED) accelerator were added to the monomer solution up to 0.2% (w/w) each. For slices, the inhibitor 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-hydroxy-TEMPO) was added up to 0.01% (w/w) from a 0.5% (w/w) stock to inhibit gelation during diffusion of the monomer solution into tissue sections. Cells or tissue slices were incubated with the monomer solution plus APS/TEMED (and 4-hydroxy-TEMPO for slices) at 4°C for one minute, 30 minutes for cultured cells, and brain slices respectively, and then transferred to a humidified 37°C incubator for two hours for gelation.
Proteinase K (New England Biolabs) was diluted 1:100 to 8 units/mL in digestion buffer (50 mM Tris (pH 8), 1 mM EDTA, 0.5% Triton X-100, 1 M NaCl) and incubated with the gels fully immersed in proteinase solution overnight at RT (this step can also be performed at 37° C for 4 hours). Digested gels were next placed in excess volumes of doubly de-ionized water for 0.25–2 hours to expand, with longer times for thicker gels. This step was repeated 3–5 times in fresh water, until the size of the expanding sample plateaued.