Sodium acetate

Colon segments were resected and soaked in saturated sodium sulfate at 4 °C. Whole-mount samples were cut and fixed in 4% paraformaldehyde fixative for 2 h at 4 °C. A fixative rinse was performed in PBS for 24 h at 4 °C. The segments were subsequently incubated and shaken in a reaction mixture of ethopropazine HCl (7.2 mg) (E5406, Sigma-Aldrich), acetylthiocholine iodide (115.6 mg) (A5751, Sigma-Aldrich), glycine (75 mg) (MP Biomedicals, Fountain Parkway Solon, OH, USA), cupric sulfate (50 mg) (NACALAI TESQUE, INC., Kyoto, Japan), sodium acetate (885 mg) (NACALAI TESQUE, INC., Kyoto, Japan) and double distilled water (100 ml) for 75 min at room temperature. Samples were rinsed six times in distilled water, and incubated for 1–2 min after adjusting the pH to 6.0 with 1.25% sodium sulfate^{34 (link)}. After six additional rinses in distilled water, photographs were obtained for each sample using a stereomicroscope (Leica). We analyzed the fields of view with high nerve fiber density using ImageJ. Nerve fibers were distinguished from the background in accordance with a preset threshold value, and the black and white areas were determined. The threshold was set at a mean filter confirming the value at which the fibers could be identified.

Methotrexate and phosphate‐buffered saline (PBS) (pH 7.4) were obtained from Sigma‐Aldrich. 1,3,7‐Trimethyluric acid (137U) was obtained from Cayman Chemical. Acetonitrile (LC‐MS‐grade) was obtained from Merck. HPLC‐grade methanol and water were obtained from FUJIFILM Wako. Acetic acid, sodium acetate, sodium hydroxide (NaOH), and hydrochloric acid (HCl) were obtained from Nacalai Tesque.

The porous α-alumina porous support (o.d. of 1.9–2.3 mm, average pore diameter of about 150 nm) was supplied by AIST (Japan). Tetraethyl orthosilicate (TEOS; >95.0%), HCl (35%), acryloyl chloride (>98.0%), acrylic acid (>98.0%), ammonium peroxodisulfate (APS; >98.0%), toluene (>95%), tetrahydrofuran (THF; >99.5%), acetic acid (>99.7%), L-lactic acid (85.0%–92.0%), phenol (>99.0%), 0.2 mol/L HCl, and 0.2 mol/L sodium hydroxide aqueous solution were purchased from FUJIFILM Wako Pure Chemical Corp., Osaka, Japan. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (>95.0%), ethanol (>99.5%), methanol (>99.8%), sodium tetraborate decahydrate (99.5–101.0%), disodium hydrogen phosphate dodecahydrate (>99.0%), and sodium dihydrogen phosphate dihydrate (99.0–102.0%) were purchased from Kanto Chemical Co., Ltd., Tokyo, Japan. 3-Aminopropyltrimethoxysilane (APTMS; >96.0%) was purchased from Tokyo Chemical Industry Co., Ltd., Tokyo, Japan. Sodium acetate (>98.0%) was purchased from Nacalai Tesque Inc., Kyoto, Japan. All chemicals were used without purification. Water was obtained from a water purification system (WG-204, Yamato Co., Tokyo, Japan).