2 2 azobis 2 2 imidazolin 2 yl propane dihydrochloride

High refractive index hydrogels were synthesised as previously described [19 (link)]. In brief, acrylamide (Sigma-Aldrich, St Louis, MO, USA) and methacrylamide monomers (Sigma-Aldrich) (3:7 ratio) were prepared in MilliQ water and cross-linked with 9% (w/v) tri(ethylene glycol) dimethylacrylate (TEDA) (Sigma-Aldrich). The composition of the mixture prior to polymerisation was 1:1 (w/w) monomers:water. The solution was heated to 60 °C for 20 min to completely dissolve the monomers, then cooled to room temperature (RT), followed by the addition of 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (0.05% (w/v)) (Wako Chemicals, Minato City, Japan), a temperature-dependent cross-linking initiator. The solution was poured into custom-made polydimethylsiloxane (PDMS) moulds, and a thin layer of mineral oil (Singer, La Vergne, TN, USA) was applied to create an anoxic environment prior to incubation at 37 °C for 1 h. The polymerised gels were removed from the moulds and immersed in PBS for 2 days, with at least 2 changes of PBS to remove non-crosslinked monomers.

For tissue clearing, we used 0.3, 1, 2, 3, and 5 mm brain slices and 1 mm-thick organ tissues. After electrophysiological recording, 0.3 mm brain slices were used to acquire biocytin-filled neuron images. Brain slices with 0.3 mm were fixed with 4% PFA for 1 h at room temperature, and others were fixed overnight at 4 °C. Samples were washed 3 times with 0.1 × PBS, incubated with polymerization buffer (A4B0; 4% acrylamide in 0.1 × PBS supplemented with 0.25% the photoinitiator 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Wako Pure Chemical, Osaka, Japan) overnight at 4 °C. Samplers were degassed for 5 min and polymerized for 3 h at 37 °C, and samples were washed in 0.1 × PBS to remove excess hydrogel. Samples were then placed in 8% SDS for 2 h at room temperature while shaking. For brain slices thicker than 1 mm, we used the ACT protocol previously described^{5 (link)}. Briefly, tissue slices were post-fixed in 4% PFA overnight at 4 °C and incubated in A4B0 solution. After polymerization, samples were placed on a tissue container in the ETC chamber and processed with following conditions: 1.5 mA, 37 °C, time depending on sample thickness (1 h for 1 mm brain slice and a 1 h increase for each 1 mm thickness, 3~6 h for 1 mm organ slices). After tissue clearing, all samples were washed in 0.1 × PBS overnight at room temperature while shaking to remove SDS.

Monomer 2-aminoethyl methacrylate hydrochloride
(Sigma 90%, AEMA) was dissolved into acetate buffer (100 mg/mL) and
stirred in an Al₂O₃/inhibitor remover mixture
thoroughly. AEMA was then extracted by centrifuging (15 min, 4025
RCF) the solids out. Chain transfer agents (CTAs) 4-cyano-4-(phenylcarbonothioylthio)pentanoic
acid (Sigma, CPA) and poly(ethylene oxide) methyl ether 2-(dodecylthiocarbonothioylthio)-2-methylpropionate
(Sigma, 6000 g/mol, PEO-CTA) were used as received. All solvents were
used as received except ultrapure deionized water, which was used
fresh from filtration equipment (18 mΩ, UHQ H₂O).
Thermal initiator 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride
(Wako Chemicals, VA-044) was recrystallized from methanol with a small
amount of UHQ H₂O. 2-Ethyl-2-thiopseudourea hydrobromide
(Sigma), sodium hydroxide (VWR aq. 0.1 M, NaOH), acetic acid (Merck
EMSURE, CH₃COOH), hydrochloric acid (Merck EMSURE fuming
or WVR aq. 1 M, HCl), sodium carbonate (Merck EMSURE, NaCO₃), sodium bicarbonate (Merck EMSURE, NaHCO₃), and sodium
acetate (Merck EMSURE, NaCH₃COO) were used as received.
Instrument grade CO₂ (Linde, pure) and N₂ (Linde,
instrument 5.0, ≥99.999%) were used as received.