Triethylamine

Glycerol, sebacic acid, triethylamine (TEA), anhydrous tetrahydrofuran (THF), bromoisobutyryl bromide (BIBB), polyethylene glycol methyl ether methacrylate (PEGMEMA), 1,1,4,7,10,10-Hexamethyltriethylenetetramine (HMTETA), copper (I) bromide, doxorubicin, triethylamine (TEA), ethyl α-bromoisobutyrate (EBIB) and α-cyclodextrin (αCD) were purchased from Sigma-Aldrich, Nucleos, Singapore. Dulbecco’s modified eagle medium (DMEM), MTT and cell culture reagents was purchased from Life Technologies (Carlsbad, CA, USA).

Palmitoylation of BSA was performed by modifying a protocol developed by the Geffard laboratory [20 (link)–22 (link)]. For this reaction, 10 mg of PA was dissolved in 10 mL of anhydrous methanol (Sigma-Aldrich) containing 100 μL of triethylamine (Sigma-Aldrich). To activate the carboxylic acid group, 200 mL of anhydrous dimethylformamide (DMF) solution containing ethylchloroformate (ETOCOCL) (Sigma-Aldrich) diluted 1/16 was added. The solution was incubated for 3 min at 4°C. Next, 200 mg FA-free BSA (Gemini Bio-Products) was dissolved in 10 mL of 1 M phosphate buffer (pH 6.8), containing 1 mM CaCl₂ and 100 μL of triethylamine (Sigma-Aldrich). The BSA solution was added to the PA solution and the mixture was stirred for 1 hr at room temperature. The BSA-PA conjugates were purified by dialysis against 150 mL of 1 mM CaCl₂ phosphate buffer (pH 6.8), containing dimethylformamide and methanol, (vol/vol/vol) overnight at room temperature (RT). The conjugate was then dialyzed against 1 M phosphate buffer (pH 6.8), containing 1 mM CaCl₂ for 24 hours at RT. The mixture was then dialyzed against PBS (pH 7.2) until precipitated phosphate became soluble. The BSA-PA was filtered to remove any remaining precipitate and the BSA-PA concentration was determined by measuring the optical density (OD) at 280 nm.

THP-Pam was synthesised using an adapted version of our previously published method^{17 (link)}. In brief, THP-NCS (10.0 mg, 10.4 µmol, CheMatech, France) was dissolved in water (400 µL) and triethylamine (42.4 µL, 30.8 mg, 0.304 mmol, Merck Life Science UK Limited, UK). Pamidronate disodium (29.0 mg, 104 µmol, synthesised as previously published^{17 (link)}) was dissolved in water (400 µL) and triethylamine (42.4 µL, 30.8 mg, 0.304 mmol, Merck Life Science UK Limited, UK). The THP-NCS solution was added to the pamidronate solution and stirred at 90 °C for 3 h. The crude solution was purified by semi-preparative HPLC: column: Agilent ZORBAX Eclipse XDB-C18 (9.4 × 250 mm, 5 μm); solvent A: water + 0.1% formic acid; solvent B: acetonitrile + 0.1% formic acid; flow rate = 4 mL min^–1; 0–5 min = 5% B, 5–40 min = 5–20% B, 40–41 min = 20–95% B, 41–45 min = 95% B, 45–46 min = 95–5% B, 46–50 min = 5% B. The identity of the product was confirmed by LC/MS, ¹H NMR and ³¹P NMR.

We coated the coverslips with a silane layer containing an allyl moiety to stabilize a polyacrylamide (PA) film introduced in the later “Tissue embedding and clearing” step^{29 (link)}. Briefly, the coverslips were cleaned in a 1:1 mixture of 37% (vol/vol) HCl and methanol for 30 min at room temperature. Then, coverslips were rinsed in deionized water three times and in 70% ethanol once. Coverslips were dried in a 70 °C oven before being immersed in 0.1% (vol/vol) triethylamine (Millipore, TX1200-11) and 0.2% (vol/vol) allytrichlorosilane (Sigma, 107778-5 G) in chloroform for 30 min at room temperature. Finally, the coverslips were washed once with chloroform and once with 100% ethanol, then dried in a 70 °C oven for 1 h and stored in a drying basin.

Seafood soy sauce (Haitian), jinbiao soy sauce (Haitian), steamed fish soy sauce (Lee Kum Kee), spicy fresh dew (Knorr), fresh shellfish sauce (Totole), Maggi fresh (Nestle), celery, carrot, onion, green onion, red pepper, ginger, garlic, coriander, dried shrimps, dried shitake mushrooms, and rock sugar were purchased from local supermarkets in Wuxi, China. Food-grade ascorbic acid and potassium sorbate were purchased from Henan Wanbang Industrial Co., Ltd. (Zhengzhou, China). The juicer was purchased from Midea Group Co., Ltd. (Foshan, China).
Methanol (chromatographically pure) and acetonitrile (chromatographically pure) were purchased from Tedia Co., Ltd. (Fairfield, Ohio, USA). Tetrahydrofuran, triethylamine, hydrochloric acid, crystalline sodium acetate, and trichloroacetic acid were of analytical grade, and water was Millipore ultrapure water. Seventeen amino acid standards, including alanine (Ala), arginine (Arg), glycine (Gly), glutamic acid (Glu), aspartic acid (Asp), methionine (Met), serine (Ser), histidine (His), leucine (Leu), phenylalanine (Phe), isoleucine (Iso), cysteine (Cys), tyrosine (Tyr), lysine (Lys), threonine (Thr), valine (Val), and proline (Pro), were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. (Shanghai, China).