Tert butanol

Following fixation in 4% PFA, samples were embedded in 1% agarose by boiling and slowly cooling down to room temperature. Samples processed with BABB were sequentially incubated in 25, 50, 80, and 100% ethanol (Merck Millipore, Burlington, MA, United States) for 8 h each at room temperature. Samples processed with 1-propanol BABB (1P-BABB) were sequentially incubated in 25, 50, 80, and 100% 1-propanol (Merck Millipore, Burlington, MA, United States) for 8 h each at room temperature. Samples processed with tert-butanol BABB (tB-BABB) were sequentially incubated in 25, 50, 80, and 100% tert-butanol (Daejung Chemical & Metals Co. Ltd., Gyeonggi-do, Korea) for 8 h each at room temperature. After alcohol dehydration, the samples were incubated in fresh solutions of either 100% ethanol, 1-propanol, or tert-butanol, as appropriate, for 12 h at room temperature. Subsequently, samples were incubated in dichloromethane (DCM; Sigma-Aldrich Inc., St. Louis, MO, United States) for 1 h at room temperature. Samples were then incubated in BABB solution [1 volume of benzyl alcohol (BA; Sigma-Aldrich Inc., St. Louis, MO, United States) to 2 volumes of benzyl benzoate (BB; Duksan Pure Chemicals, Gyeonggi-do, Korea)] for 1–2 days until they achieved optical transparency.

Korean red pine (Pinus densiflora S. et Z.) was obtained from the Experimental Forest of Kangwon National University (Chuncheon, Korea). The degreased wood powder was prepared using an ethanol/benzene (1/2, v/v) solution in a Soxhlet extractor operating at 90 °C for 6 h. AL, CaCl₂, ChCl, LA, sodium chlorite, acetic acid, 50% NaOH solution, tert-butanol, and sulfuric acid were purchased from Daejung Chemical & Metals Co., Ltd. (Siheung, Korea) and used without further purification. Commercial PCNFs were supplied by Cellulose Lab, Co., Ltd. (Fredericton, NB, Canada).

Naringin (95%) and ethyl acetate (99.8%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Novozym 435 (immobilized Candida antarctica lipase B), Lipozyme TL IM (immobilized Thermomyces lanuginosus), and Lipozyme RM IM (immobilized Rhizomucor miehei) were purchased from Novo Nordisk Bioindustry (Bagsvaerd, Denmark). Dimethyl sulfoxide (DMSO) (99.5%), 1,4-dioxane (99.5%), acetone (99.5%), tert-butanol (99.5%), tert-amyl alcohol (99%), 1,2-dichloroethane (99%), isooctane (98%), oleic acid (99%), and molecular sieves (3-Å) were purchased from Dae-Jung Chemical and Metals Co. Ltd. (Gyunggido, Korea). Acetonitrile (99.5%) and tetrahydrofuran (THF) (99.5%) were purchased from Junsei Chemical Co. Ltd. (Tokyo, Japan). Methanol, water, and acetic acid (HPLC grade) were purchased from J.T. Baker (Phillipsburg, NJ, USA).