2 2 2 nitrilotriethanol

Paraformaldehyde, formamide, fructose, urea, 2,2′,2″-nitrilotriethanol, and sucrose were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Polyethyleneglycol (PEG; molecular weight: 4,000) and Triton X-100 were purchased from Nacalai Tesque (Kyoto, Japan). N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine and α-thioglycerol were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Sigma-Aldrich Co. Llc. (St. Louis, MO, USA). Tetramethylrhodamine (TMR)-labeled dextran (molecular weight: 10,000; lysine fixable) was purchased from Molecular Probes, Inc. (Eugene, OR, USA).

A 12 × 12 mm square segment was obtained from a full-thickness skin sample. After stretching tissues in all directions 1.25-fold using the biaxial extension device, samples were immersed and fixed using 4% paraformaldehyde for 24 h in their stretched state. Large full-thickness skin samples were fixed in an unstretched state. To maximize the penetration of the laser beam used in microscopic observation, the tissue clearing (CUBIC) method was used^{27 (link),28 (link)}. Briefly, tissues were immersed in Reagent-1 at 37 °C for 1 week, which consists of 25 wt% urea (Nacalai Tesque Inc., Kyoto, Japan), 25 wt% N,N,N′,N′-tetrakis(2-hydroxypropyl)ethylenediamine (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan), and 15 wt% polyethylene glycol mono-p-isooctylphenyl ether/Triton X-100 (Nacalai Tesque Inc.)) and then immersed in Reagent-2 for 3 days, which consists of 50 wt% sucrose (Nacalai Tesque Inc.), 25 wt% urea, 10 wt% 2,2′,2′′-nitrilotriethanol (FUJIFILM Wako Pure Chemical Co., Ltd., Tokyo, Japan), and 0.1% (v/v) Triton X-100).

Paraformaldehyde (PFA) (CAS 30525-89-4), urea (CAS 57-13-6), sucrose (CAS 57-50-1), and 2,2’,2’’-nitrilotriethanol (CAS 102-71-6) were purchased from Wako Pure Chemical Industries, Ltd. Polyethylene glycol mono-p-isooctylphenyl ether (Triton X-100; CAS 9002-93-1) was purchased from Nacalai Tesque. N,N,N’,N’-tetrakis (2-hydroxypropyl) ethylenediamine (CAS 102-60-3) was purchased from Tokyo Chemical Industry Co., Ltd.